WO2023156505A1 - Trimer stabilizing hiv envelope protein mutations r304v, n302m and t320l - Google Patents

Abstract

The invention relates to recombinant HIV envelope proteins that have improved folding as compared to certain previously described HIV envelope trimers. This improved folding leads to higher quality trimers with reduced non-neutralising antibodies (non-nAbs). Also provided are particles displaying the HIV envelope proteins, nucleic acid molecules and vectors encoding the HIV envelope proteins, as well as compositions containing the HIV envelope proteins, particles, nucleic acid, or vectors.

Description

TRIMER STABILIZING HIV ENVELOPE PROTEIN MUTATIONS R304V, N302M AND T320L

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus (HIV) affects millions of people worldwide, and the prevention of HIV through an efficacious vaccine remains a very high priority, even in an era of widespread antiretroviral treatment. Antigenic diversity between different strains and clades of the HIV virus renders it difficult to develop vaccines with broad efficacy. HIV-1 is the most common and pathogenic strain of the virus, with more than 90% of HIV/AIDS cases deriving from infection with HIV-1 group M. The M group is subdivided further into clades or subtypes, of which clade C is the largest. An efficacious vaccine ideally would be capable of eliciting both potent cellular responses and broadly neutralizing antibodies capable of neutralizing HIV-1 strains from different clades.

The envelope protein spike (Env) on the HIV surface is composed of a trimer of heterodimers of glycoproteins gpl20 and gp41. The precursor protein gpl60 is cleaved by furin into gpl20, which is the head of the spike and contains the CD4 receptor binding site as well as the large hypervariable loops (VI to V5), and gp41, which is the membrane-anchored stem of the envelope protein spike. Like other class I fusion proteins, gp41 contains an N- terminal fusion peptide (FP), a C-terminal transmembrane (TM) domain, and a cytoplasmic domain. Membrane fusion between HIV and target cell membranes requires a series of conformational changes in the envelope protein. HIV vaccines can be developed based upon the envelope protein.

However, various factors make the development of an HIV vaccine based upon the envelope protein challenging, including the high genetic variability of HIV-1, the dense carbohydrate coat of the envelope protein, and the relatively dynamic and labile nature of the envelope protein spike structure. The limited number of properly folded, functional Env trimers present on the surface of each virion is thought to be one of the many ways HIV is able to evade the immune system. Unprocessed Env protein that is not cleaved into gpl20- gp41 heterodimers is non-functional, and as a result exposes non-neutralizing epitopes. These forms of Env are widely thought to act as decoys to the immune system by displaying epitopes (cluster I and II) on gp41 that are usually occluded by the trimer in a native closed trimeric conformation and therefore, antibodies elicited to these sites are able to bind but not neutralize HIV. Moreover, the shedding of gp!20 from Env leads to the presence of circulating gp120 monomers that expose immunodominant sites on both gp120 and gp41 and elicit non-neutralising antibodies (non-nAbs). WO 2019/079337 discloses that HIV-1 Env protein is stabilized by the presence of mutations N302M and T320L. Steichen et al. (2016, Immunity 45: 483–496) disclose that the gp120 variant MD16, which comprises the three mutations F223W, R304V, and A319Y, reduced binding to V3 non-neutralizing antibodies. For vaccine development, it is preferred to use envelope proteins that can induce bNAbs and that elicit the least amount of non-nAbs. Accordingly, there is a need for trimers of HIV envelope proteins that display good binding with bNAbs, and decreased binding to non-bNAbs. Preferably, such trimers decrease the binding of non-bNAbs without affecting trimer formation, improved trimer yield, and/or improved trimer stability. SUMMARY OF THE INVENTION The invention relates to recombinant HIV envelope proteins that have improved folding as compared to certain previously described HIV envelope trimers. This improved folding leads to higher quality trimers with reduced non-bNAb. The invention also relates to isolated nucleic acid molecules and vectors encoding the recombinant HIV envelope proteins, cells comprising the same, and compositions of the recombinant HIV envelope protein, nucleic acid molecule, vector, and/or cells. In a general aspect, the invention provides for a recombinant human immunodeficiency virus (HIV) envelope (Env) protein, comprising the following amino acid residues: Met at position 302, Val at position 304, and Leu at position 320, wherein the numbering of the positions is according to the numbering in gp160 of HIV-1 isolate HXB2. In certain embodiments, such HIV Env proteins further comprise one or more mutations that increase trimer yield and/or stabilize trimers, as indicated herein. In certain embodiments, the recombinant HIV Env protein of the invention further comprises a) Cys at positions 501 and 605, or b) Pro at position 559 or c) Cys at positions 501 and 605 and Pro at position 559. In certain embodiments, a recombinant HIV Env protein of the invention further comprises one or more of the following amino acid residues at the indicated positions: (i) Pro at position 556 (ii) Phe, Ile, Met, or Trp, preferably Ile, at position 655; (iii) Phe, Leu, Met, or Trp, preferably Phe, at position 651; (iv) Asn or Gln, preferably Asn, at position 535; (v) Val, Ile or Ala, preferably Val, at position 589; (vi) Gln, Glu, Ile, Met, Val, Trp, or Phe, preferably Gln or Glu, at position 588; (vii) Val, Ile, Phe, Met, Ala, and Leu, preferably Val at position 658. (viii) 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; (ix) Pro at position 329; (x) Asp at position 586; (xi) Ile at position 204; (xii) Phe or Trp, preferably Phe, at position 573 (xiii) Lys at position 64 or Arg at position 66 or Lys at position 64 and Arg at position 66; (xiv) Trp at position 316; (xv) Pro at position 558 or at both positions 556 and 558; (xvi) replacement of the loop at amino acid positions 548-568 (HR1-loop) by a loop having 7-10 amino acids, preferably a loop of 8 amino acids, for example having a sequence chosen from any one of (SEQ ID NOs: 9-14); (xvii) or Arg at position 519, or Arg at position 520, or Arg at both positions 519 and 520; (xviii) His at position 108; (xix) His at position 538, (xx) Trp, Phe, Met, or Leu, preferably Trp at position 650; (xxi) a mutation in a furin cleavage sequence of the HIV Env protein, preferably a replacement at positions 508-511 by RRRRRR (SEQ ID NO: 6); and/or (xxii) Cys at both positions 201 and 433. In certain embodiments, the recombinant HIV Env protein of the invention comprises Pro at position 556, Ile at position 655, Phe at position 651, Asn at position 535, Val at position 589, Gln or Glu at position 588, and Val at position 658. In certain embodiments, a recombinant HIV Env protein according to the invention is from a clade A, B, or C HIV. In one embodiment, the recombinant HIV Env protein is a gp140 protein based on the ectodomain or a gp160 protein, or an Env protein having a truncation in the cytoplasmic region. In another general aspect, the invention relates to a trimeric complex comprising a noncovalent oligomer of three of any of the recombinant HIV Env proteins described herein.

In another general aspect, the invention relates to a particle, e.g. a liposome or a nanoparticle, e.g. a self-assembling nanoparticle, displaying on its surface a recombinant HIV Env protein of the invention, or a trimeric complex of the invention.

In another general aspect, the invention relates to an isolated nucleic acid molecule encoding a recombinant HIV Env protein of the invention.

In another general aspect, the invention relates to vectors comprising the 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 adenovirus vector.

In another embodiment, expression is accomplished by either DNA, RNA, modified RNA, circular RNA or replicating RNA.

Another general aspect relates to a host cell comprising the isolated nucleic acid molecule or vector encoding the recombinant HIV Env protein of the invention. Such host cells can be used for recombinant protein production, recombinant protein expression, or the production of viral particles, such as recombinant adenovirus.

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

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

In another general aspect, the invention relates to a method of improving the trimer folding of an HIV Env protein, the method comprising introducing the substitution of Asn at position 302 by Met, Arg at position 304 by Vai, and Thr at position 320 by Leu, into a parent HIV Env protein, wherein the numbering of the positions is according to the numbering in gp!60 of HIV-1 isolate HXB2. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A and IB show a schematic representation of the structure of HIV envelope (Env) proteins. FIG. 1 A shows a full-length HIV Env protein and FIG. IB shows a soluble HIV Env protein containing the so-called SOSIP mutations and a C-terminal truncation beginning at residue 664 according to the numbering in gpl60 of HIV-1 isolate HXB2.

FIGS. 2A and 2B. N302M, T320L and R304V decrease non-bNAb binding to

ConB_SOSIP. FIG. 1 A Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for HIV Env ConB SOSIP variants. FIG. 2B AlphaLISA binding of the cell culture supernatants with HIV Env-specific bNAbs and non-bNAbs to ConB SOSIP variants.

FIG. 3. N302M, T320L and R304V increases trimer yield of ConB SOSIP comprising additional mutations. Analytical SEC with Expi293F cell culture supernatants after in duplo transfection with plasmids coding for HIV Env ConB SOSIP + L556P + K655I + N651F + I535N + D589V + K588E + Q658V (ConB_SOSIP+7mut) and ConB SOSIP + L556P + K655I + N651F + I535N + D589V + K588E + Q658V + N302M + T320L + R30V (ConB_SOSIP+7mut+N302M+T320L+R30V).

FIG. 4. Binding of bNAbs and non-bNAbs to ConB SOSIP+N302M+T320L+R304V.

AlphaLISA counts in duplo are shown: after incubation of 5 minutes at 80°C bNAb binding (to conformational epitopes) is lost, whereas non-bNAb binding is gained.

Fig. 5A-D. N302M, T320L and R304V decrease non-bNAb binding to ConC_SOSIP having additional mutations. Fig 5A. 2MUT (N302M, T320L; Chuang et al., 2017, Chuang et al. 2020) and addition of A329P (3MUT: A329P, N302M, T320L; Chuang et al. 2020), 2xG (Gly at position 569 and 636; as described in Guenaga et al., 2017). L568D and R304V (Steichen et al., 2016). Various combinations of mutations were made. Black boxes indicate the presence of the mutations. Compared to the ConC_SOSIP_7mut (ConC SOSIP + L556P + K655I + N651F + I535N + D589V + K588E + Q658V), the stabilized designs improve bNAb binding (FIG. 5B), reduce non-bNAb binding (FIG. 5C) measured in AlphaLISA, and increase trimer content (FIG. 5D). The variant containing N302M, T320L, R304V, A329P, 2xG, L586D shows that 2xG is not required for lower non-bNAb binding, L586D improves bNAb binding but increases non-bNAb binding and that R304V reduces non-bNAb binding.

FIGS 6A-D. N302M, T320L and R304V decrease non-bNAb binding to a repaired and stabilized Env of HIV strain TV1.29 (RnS_sl_TV1.29; SEQ ID NO: 21) FIG 6A: Various combinations of mutations were made [(N302M, T320L), (A329P, N302M, T320L, Chuang et al. 2020), (A329P, N302M, T320L + 2xG), (N302M, T320L, L568D) (N302M, T320L, R304V, 2xG, L568D)]. Black boxes indicate the presence of the mutations. Compared to the RnS Clade C strain TV1.29, the stabilized designs improve bNAb binding (FIG.6B), reduce non-bNAb binding (FIG.6C) measured in AlphaLISA, and increase trimer content (FIG. 6D). The variant containing N302M, T320L, R304V, A329P, 2xG, L586D shows optimal antigenicity. The 2xG is not required for lower non-bNAb binding. L586D increases non- bNAb binding. R304V reduces non-bNAb binding.

FIGS 7A-7B. N302M, T320L and R304V decrease non-bNAb binding to full-length ConC Env in which SOS and DS were removed. FIG.7A: Median fluorescence intensities measured by flow cytometry with (ConC_SOSIP_A204I + V295N + K655I + N651F + I535N + I573F + D589V + K588E + Q658V + R696V + DS (= I201C + A433C) + furin cleavage site RRRRRR/REKR replaced with GGGGSGGGGS) and (ConC_I559P + A204I + V295N + K655I + N651F + I535N + D589V + K588E + R696V + _N302M_T320L_R304V + furin cleavage site RRRRRR/REKR replaced with GGGGSGGGGS) expressed in adherent HEK293 cells. Measurement was done in duplo. FIG. 7B: A FACS plot of the non-bNAb 14E binding showing the decrease of binding of 14E in APC-A and fluorescent positive cells.

FIGS. 8A-8D. N302M, T320L and R304V decrease non-bNAb binding (F105 and 14E) to full-length membrane bound ConC Env on virus-like particles. AlphaLISA on VLPs with membrane bound ConC Env. The black line represents the ConC backbone without N302M, T320L and R304V and the dotted black line with the N302M, T320L and R304V mutations. The grey line represents the mock. FIGS. 9A-C. N302M, T320L and R304V decrease non-bNAb binding to full-length

ConC Env. FIGS 9A-9B: Median fluorescence intensities measured by flow cytometry with full-length ConC (backbone is SEQ ID NO: 18), backbone comprising N302M and T320L (2 MUT) and backbone comprising N302M, T320L and R304V (3 MUT) expressed in adherent HEK293 cells, of bNAb binding and non-bNAb binding, respectively. Measurement was done in duplo. FIG. 9C ratio of bNAb versus non-bNAb binding.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms cited herein have the meanings as set in the specification. All patents published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

Throughout this description and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.

When used herein “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of “comprising”, “containing”, “including”, and “having”, whenever used herein in the context of an aspect or embodiment of the invention can be replaced with the term “consisting of’ or “consisting essentially of’ to vary scopes of the disclosure. As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or”, a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”

Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

Amino acids are referenced throughout the disclosure. There are twenty naturally occurring amino acids, as well as many non-naturally occurring amino acids. Each known amino acid, including both natural and non-natural amino acids, has a full name, an abbreviated one letter code, and an abbreviated three letter code, all of which are well known to those of ordinary skill in the art. For example, the three and one letter abbreviated codes used for the twenty naturally occurring amino acids are as follows: alanine (Ala; A), arginine (Arg; R), aspartic acid (Asp; D), asparagine (Asn; N), cysteine (Cys; C), glycine (Gly; G), glutamic acid (Glu; E), glutamine (Gin; Q), histidine (His; H), isoleucine (He; 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 (Vai; V). Amino acids can be referred to by their full name, one letter abbreviated code, or three letter abbreviated code.

Unless the context clearly dictates otherwise, the numbering of positions in the amino acid sequence of an HIV envelope protein as used herein is according to the numbering in gpl60 of HIV-1 isolate HXB2 as is conventional in the field of HIV Env proteins. The gpl60 of HIV-1 isolate HXB2 has the amino acid sequence shown in SEQ ID NO: 1. Alignment of an HIV Env sequence of interest with this sequence can be used to find the corresponding amino acid numbering in the sequence of interest. It is understood herein that when reference is made to amino acid substitutions at position T320 according to the numbering in gp!60 of HIV-1 isolate HXB2, the position corresponding to position 320 in the HXB2 amino acid sequence of SEQ ID NO: 1 is meant, even though the HXB2 amino acid sequence has an isoleucine residue as opposed to the consensus threonine residue at position 320. Hence, a reference at position T320 according to the numbering in gpl60 of HIV-1 isolate HXB2 does not refer to threonine residue at position 319 in SEQ ID NO: 1.

The term “percent (%) sequence identity” or “%identity” describes the number of matches (“hits”) of identical amino acids of two or more aligned amino acid sequences as compared to the number of amino acid residues making up the overall length of the amino acid sequences. In other terms, using an alignment, for two or more sequences the percentage of amino acid residues that are the same (e.g. 95%, 97% or 98% identity) may be determined, when the sequences are compared and aligned for maximum correspondence as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. The sequences which are compared to determine sequence identity may thus differ by substitution(s), addition(s) or deletion(s) of amino acids. Suitable programs for aligning protein sequences are known to the skilled person. The percentage sequence identity of protein sequences can, for example, be determined with programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).

A ‘corresponding position’ in a HIV Env protein refers to position of the amino acid residue when at least two HIV Env sequences are aligned. Unless otherwise indicated, amino acid position numbering for these purposes is according to numbering in gpl60 of HIV-1 isolate HXB2, as customary in the field.

The “mutations according to the invention” as used herein are substitutions of the amino acids at positions 302 by methionine (Met), 304 by valine (Vai) and 320 by leucine (Leu). The HIV Env proteins comprising Met at position 302, Vai at position 304 and Leu at position 320 can optionally further comprise a) Cys at positions 501 and 605, or (b) Pro at position 559, or preferably (c) Cys at positions 501 and 605 and Pro at position 559, wherein the numbering of the positions is according to the numbering in gpl60 of HIV-1 isolate HXB2. In further embodiments, the HIV Env proteins comprising Met at position 302, Vai at position 304 and Leu at position 320 can optionally further comprise one or more additional stabilizing mutations.

Additional “stabilizing mutation” as used herein is a mutation as described herein in any of entries (i)-(xxii) of Table 1, which increases the percentage of trimer and/or the trimer yield (which can for instance be measured according to AlphaLISA or size exclusion chromatography (SEC) assays, e.g. analytical SEC assays described herein, or SEC-MALS as described e.g. in WO 2019/016062) of an HIV Env protein as compared to a parent molecule when the mutation is introduced by substitution of the corresponding amino acid in said parent molecule (see e.g. WO 2019/016062). The amino acids resulting from such stabilizing mutations typically are rarely, if at all, found in Env proteins of wild-type HIV isolates.

The terms ‘natural’ or ‘wild-type’ are used interchangeably herein when referring to HIV strains (or Env proteins therefrom), and refer to HIV strains (or Env proteins therefrom) as occurring in nature, e.g. such as in HIV-infected patients.

The invention generally relates to recombinant HIV envelope (Env) proteins comprising the amino acid substitutions N302M, R304V, and T320L in the envelope protein sequence. Env proteins comprising mutations N302M and T320L have previously been described to decrease the binding of non-bNAbs. However it has been suprisingly observed that the addition of the mutation R304V synergistically enhances this effect. It has been observed that introducing Met at position 302, Vai at position 304 and Leu at position 320 into the sequence of an HIV envelope protein stabilizes the apex in a closed conformation, which results in a decrease in non-bNAb binding. The non-bNAbs bind either misfolded, non-native Envs or the highly variable loop 3. It is thus a positive attribute for HIV Env variants when binding of one or more non-bNAbs decreases, as compared to a parent Env molecule. Assessment of the binding of bNAbs can be measured for example by an AlphaLISA assay as described herein.

Additionally, it has been observed that the amino acid substitutions as described herein stabilize the trimer form of the envelope protein which can result in an increased trimer yield and a decreased monomer yield. This can for instance be measured using trimer- specific antibodies, melting temperature, size exclusion chromatography, and binding to antibodies that bind to correctly folded (stable trimeric) or alternatively to incorrectly folded (non-stable or non-trimeric) Env protein, and increased trimer percentage and/or trimer yield is considered indicative of stable, native, correctly folded Env protein.

Human immunodeficiency virus (HIV) is a member of the genus Lentivirinae, which is part of the family of Retroviridae. Two species of HIV infect humans: HIV-1 and HIV-2. HIV-1 is the most common strain of HIV, and is known to be more pathogenic than HIV-2. As used herein, the terms “human immunodeficiency virus” and “HIV” refer to, but are not limited to, HIV-1 and HIV-2. In preferred embodiments, HIV refers to HIV-1.

HIV is categorized into multiple clades with a high degree of genetic divergence. As used herein, the term “HIV clade” or “HIV subtype” refers to related human immunodeficiency viruses classified according to their degree of genetic similarity. The largest group of HIV-1 isolates is called Group M (major strains) and consists of at least twelve clades, A through L.

In one general aspect, the invention relates to a recombinant HIV envelope (Env) protein. The term “recombinant” when used with reference to a protein refers to a protein that is produced by a recombinant technique or by chemical synthesis in vitro. According to embodiments of the invention, a “recombinant” protein has an artificial amino acid sequence in that it contains at least one sequence element (e.g., amino acid substitution, deletion, addition, sequence replacement, etc.) that is not found in the corresponding naturally occurring sequence. Preferably, a “recombinant” protein is a non-naturally occurring HIV envelope protein that is optimized to induce an immune response or produce an immunity against one or more naturally occurring HIV strains.

The terms “HIV envelope protein,” “HIV Env,” and “HIV Env protein” refer to a protein, or a fragment or derivative thereof, that is in nature expressed on the envelope of the HIV virion and enables an HIV to target and attach to the plasma membrane of HIV infected cells. The terms “envelope” and “Env” are used interchangeably throughout the disclosure. The HIV env gene encodes the precursor protein gpl60, which is proteolytically cleaved into the two mature envelope glycoproteins gpl20 and gp41. The cleavage reaction is mediated by a host cell protease, furin (or by furin-like proteases), at a sequence motif highly conserved in retroviral envelope glycoprotein precursors. More specifically, gpl60 trimerizes to (gp 160)3 and then undergoes cleavage into the two noncovalently associated mature glycoproteins gpl20 and gp41. Viral entry is subsequently mediated by a trimer of gpl20/gp41 heterodimers. Gpl20 is the receptor binding fragment, and binds to the CD4 receptor (and the co-receptor) on a target cell that has such a receptor, such as, e.g., a T-helper cell. Gp41, which is non-covalently bound to gpl20, is the fusion fragment and provides the second step by which HIV enters the cell. Gp41 is originally buried within the viral envelope, but when gpl20 binds to a CD4 receptor and co-receptor, gpl20 changes its conformation causing gp41 to become exposed, where it can assist in fusion with the host cell. Gpl40 is the ectodomain of gpl60.

According to embodiments of the invention, an “HIV envelope (Env) protein” can be a gpl60 or gpl40 protein, or combinations, fusions, truncations, or derivatives thereof. For example, an “HIV envelope protein” can include a gpl20 protein noncovalently associated with a gp41 protein. An “HIV envelope protein” can also be a truncated HIV envelope protein including, but not limited to, envelope proteins comprising a C-terminal truncation in the ectodomain (i.e. the domain that extends into the extracellular space), a truncation in the gp41, such as a truncation in the ectodomain of gp41, in the transmembrane domain of gp41, or a truncation in the cytoplasmic domain of gp41. An HIV envelope protein can also be a gpl40, corresponding to the gpl60 ectodomain, or an extended or truncated version of gpl40. Expression of gpl40 proteins has been described in several publications (e.g. Zhang et al., 2001; Sanders et al., 2002; Harris et al., 2011), and the protein can also be ordered from service providers, in different variants e.g. based on different HIV strains. A gpl40 protein according to the invention can have a cleavage site mutation so that the gpl20 domain and gp41 ectodomain are not cleaved and covalently linked, or alternatively the gpl20 domain and gp41 ectodomain can be cleaved and covalently linked, e.g. by a disulfide bridge (such as for instance in the SOSIP variants). An “HIV envelope protein” can further be a derivative of a naturally occurring HIV envelope protein having sequence mutations, e.g., in the furin cleavage sites, and/or so-called SOSIP mutations. An HIV envelope protein according to the invention can also have a cleavage site so that the gpl20 and gp41 ectodomain can be non- covalently linked.

In preferred embodiments of the invention, the HIV Env protein is a gpl40 protein or a gpl60 protein, and more preferably a gpl40 protein. In other preferred embodiments the Env protein is truncated, e.g. by deletion of the residues after the 7th residue of the cytoplasmic region as compared to a natural Env protein.

According to embodiments of the invention, an “HIV envelope protein” can be a trimer or a monomer, and is preferably a trimer. The trimer can be a homotrimer (e.g., trimers comprising three identical polypeptide units) or a heterotrimer (e.g., trimers comprising three polypeptide units that are not all identical). Preferably, the trimer is a homotrimer. In case of a cleaved gpl40 or gpl60, it is a trimer of polypeptide units that are gpl20-gp41 dimers, and in case all three of these dimers are the same, this is considered a homotrimer.

An “HIV envelope protein” can be a soluble protein, or a membrane bound protein. Membrane bound envelope proteins typically comprise a transmembrane domain, such as in the full length HIV envelope protein comprising a transmembrane domain (TM). Membrane bound proteins can have a cytoplasmic domain, but do not require a cytoplasmic domain to be membrane bound. Soluble envelope proteins comprise at least a partial or a complete deletion of the transmembrane domain. For instance, the C-terminal end of a full length HIV envelope protein can be truncated to delete the transmembrane domain, thereby producing a soluble protein (see e.g. Fig 1A and IB in WO 2019/016062 for schematic representations of full length and truncated soluble HIV Env proteins, respectively). However, the HIV envelope protein can still be soluble with shorter truncations and alternative truncation positions to those shown in FIG. IB of WO 2019/016062. Truncation can be done at various positions, and non-limiting examples are after amino acid 664, 655, 683, etc. which all result in soluble protein. A membrane-bound Env protein according to the invention may comprise a complete or a partial C-terminal domain (e.g. by partial deletion of the C-terminal cytoplasmic domain, e.g. in certain embodiments after the 7th residue of the cytoplasmic region) as compared to a native Env protein. It will be clear to the skilled person that the deletion in the cytoplasmic region can also be from another than the 7th residue of the cytoplasmic domain, e.g. after the 1^st, 2^nd, 3^rd, 4^th, 5^th, 6^th, 8^th, 9^th, 10^th, or any later residue of the cytoplasmic domain.

A signal peptide is typically present at the N-terminus of the HIV Env protein when expressed, but is cleaved off by signal peptidase and thus is not present in the mature protein. The signal peptide can be interchanged with other signal sequences, and two non-limiting examples of signal peptides are provided herein in SEQ ID NOs: 7 and 8.

According to embodiments of the invention, the HIV envelope protein, e.g., gpl60, or gpl40, can 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 in ‘circulating recombinant forms’ or CRFs derived from recombination between viruses of different subtypes, e.g BC, AE, AG, BE, BF, ADG, etc). The HIV envelope protein sequence can be a naturally occurring sequence, a mosaic sequence, a consensus sequence, a synthetic sequence, or any derivative or fragment thereof. A “mosaic sequence” contains multiple epitopes derived from at least three HIV envelope sequences of one or more HIV clades, and may be designed by algorithms that optimize the coverage of T-cell epitopes. Examples of sequences of mosaic HIV envelope proteins include those described in, e.g., Barouch et al, Nat Med 2010, 16: 319-323; and WO 2010/059732, such as for instance those shown in SEQ ID NOs: 8 and 9 of WO 2010/059732. As used herein “consensus sequence” means an artificial sequence of amino acids based on an alignment of amino acid sequences of homologous proteins, e.g. as determined by an alignment (e.g. using Clustal Omega) of amino acid sequences of homologous proteins. It is the calculated order of most frequent amino acid residues, found at each position in a sequence alignment, based upon sequences of Env from for example at least 1000 natural HIV isolates. A “synthetic sequence” is a non-naturally occurring HIV envelope protein that is optimized to induce an immune response or produce immunity against more than one naturally occurring HIV strains. Mosaic HIV envelope proteins are non-limiting examples of synthetic HIV envelope proteins. In preferred embodiments of the invention, the parent HIV Env protein is a consensus Env protein, or a synthetic Env protein. In the parent Env protein, a mutation is introduced to result in amino acid Met at position 302, Vai at position 304 and Leu at position 320. Optionally, such HIV Env protein may further have at least one of the indicated amino acids at the indicated positions (i)-(xxiii) described herein in Table 1. For example, in certain embodiments, the invention provides for Env proteins having Met at position 302, Vai at position 304 and Leu at position 320 and further having SOSIP (e.g. the indicated amino acids at position xxiii in Table 1 and as described below) and/or preferably having further at least one, two, three, four, five, six or seven of the indicated amino acid residues at the indicated positions (i)-(vii) as described in Table 1.

In certain embodiments of the invention, an HIV envelope protein, whether a naturally occurring sequence, mosaic sequence, consensus sequence, synthetic sequence etc., comprises additional sequence mutations e.g., in the furin cleavage sites, and/or at least one of the amino acids at the indicated positions (i)-(xxiii) described herein in Table 1.

In some embodiments of the invention, an HIV envelope protein of the invention has further mutations and is a “SOSIP mutant HIV Env protein.” The so-called SOSIP mutations are trimer stabilizing mutations that include the ‘SOS mutations’ (Cys residues at positions 501 and 605, which results in the introduction of a possible disulfide bridge between the newly created cysteine residues) and the ‘IP mutation’ (Pro residue at position 559). According to certain embodiments of the invention, a mutant Env protein comprises 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. A SOSIP mutant HIV Env protein can further comprise other sequence mutations, e.g., in the furin cleavage site. In addition, in certain embodiments it is possible to further add mutations such that the Env protein comprises Pro at position 556 or position 558 or at positions 556 and 558, which were found to be capable of acting not only as alternatives to Pro at position 559 in a SOSIP variant, but also as additional mutations that could further improve trimer formation of a SOSIP variant that already has Pro at position 559.

In certain preferred embodiments of the invention, a SOSIP mutant HIV Env protein comprises Cys at positions 501 and 605, and Pro at position 559.

In certain embodiments, an HIV envelope protein of the invention further comprises a mutation in the furin cleavage site. The mutation in the furin cleavage sequence can be an amino acid substitution, deletion, insertion, or replacement of one sequence with another, or replacement with a linker amino acid sequence. Preferably in the present invention, mutating the furin cleavage site can be used to optimize the cleavage site, so that furin cleavage is improved over wild-type, for instance by a replacement of the sequence at residues 508-511 with RRRRRR (SEQ ID NO: 6) [i.e. replacement of a typical amino acid sequence (e.g. EK) at positions 509-510 with four arginine residues (i.e. two replacements and two additions), while at positions 508 and 511, there are already arginine residues present in most HIV Env proteins, so these typically do not need to be replaced, but since the end result in literature is often referred to as amino acid sequence RRRRRR, we kept this nomenclature herein]. Other mutations that improve furin-cleavage are known and can also be used. Alternatively, it is possible to replace the furin cleavage site with a linker, so that furin cleavage is no longer necessary but the protein will adopt a native-like conformation (e.g. described in (Sharma et al, 2015) and (Georgiev et al, 2015)).

In particular embodiments of the invention, an HIV envelope protein of the invention further comprises both the so-called SOSIP mutations (Cys at positions 501 and 605, and Pro at position 559) and a sequence mutation in the furin cleavage site, preferably a replacement of the sequence at residues 508-511 with RRRRRR (SEQ ID NO: 6). In certain preferred embodiments, the HIV Env comprises both the indicated SOSIP and furin cleavage site mutations, and in addition further comprises a Pro residue at position 556 or 558, most preferably at both positions 556 and 558.

In certain embodiments of the invention, the amino acid sequence of the HIV envelope protein is a consensus sequence, such as an HIV envelope clade C consensus or an HIV envelope clade B consensus.

Exemplary HIV envelope proteins that can be used in the invention include HIV envelope clade C consensus (SEQ ID NO: 2) and HIV envelope clade B consensus (SEQ ID NO: 4). These HIV envelope clade C and clade B consensus sequences can comprise additional mutations that, e.g., enhance stability and/or trimer formation, such as for instance the so-called SOSIP mutations and/or a sequence mutation in the furin cleavage site as described above, such as for instance in the ConC SOSIP sequence shown in SEQ ID NO: 3 and the ConB SOSIP sequence shown in SEQ ID NO: 5.

Other non-limiting examples of preferred HIV envelope protein sequences that can be used in the invention (as ‘background’ or ‘parent’ molecule, wherein then position 302 is mutated into Met, position 304 is mutated into Vai and position 320 is mutated into Leu) include synthetic HIV Env proteins, optionally having further SOSIP, furin cleavage site mutations and/or the mutations (i)-(vii) described herein in Table 1. Further non-limiting examples are mosaic HIV envelope proteins.

In certain preferred embodiments, the parent molecule is a synthetic HIV envelope further comprising Pro at position 556, He at position 655, Phe at position 651, Asn at position 535, Vai at position 589, Gin or Glu at position 588, and Vai at position 658, SOSIP and furin cleavage site mutations. In certain embodiments, the parent molecule is a wild-type HIV Env protein. Such a parent molecule may optionally further have SOSIP, furin cleavage site mutations as described above and/or the mutations (i)-(vii) described herein in Table 1.

In certain preferred embodiments, the parent molecule is a wild-type HIV Env protein further comprising Pro at position 556, He at position 655, Phe at position 651, Asn at position 535, Vai at position 589, Gin or Glu at position 588, and Vai at position 658, SOSIP and furin cleavage site mutations.

Mutations resulting in the amino acid at positions 302, 304 and 320 being replaced with respectively amino acid Met, Vai, and Leu, optionally further with the indicated amino acids at positions (i)-(xxii) described in Table 1, can also be used in HIV Env proteins wherein no SOSIP mutations are present (e.g. in Env consensus sequences or in Env proteins from wild-type HIV isolates). Thus, in certain embodiments, an HIV Env protein according to the invention does not include any of the SOSIP mutations.

A recombinant HIV envelope protein according to embodiments of the invention comprises an HIV envelope protein having certain amino acid residue(s) at specified positions in the amino acid sequence of an HIV envelope protein. In particular, it was shown that positions 302, 304 and 320 could be mutated to respectively amino acids Met, Vai, and Leu to decrease the binding of non-bNAbs wherein the numbering of the positions is according to the numbering in gpl60 of HIV-1 isolate HXB2. In addition, in optional embodiments, a number of positions in the envelope protein are indicated, as well as the particular amino acid residues to be desirable at one or more or each of the identified positions, in Table 1, wherein the numbering of the positions is according to the numbering in gpl60 of HIV-1 isolate HXB2. An HIV Env protein according to the invention has Met at position 302, Vai at position 304 and Leu at position 320, and optionally has the specified amino acid residue(s) in at least one of the indicated positions (i)-(xxiii) as provided in Table 1. Table 1: Additional Desirable Amino Acids at Indicated Positions in the Recombinant

HIV Env Proteins According to Certain Embodiments

¹ According to the numbering in gp!60 of HIV-1 isolate HXB2

The amino acid sequence of the HIV envelope protein into which the Met at position 302, the Vai at position 304 and the Leu at position 320, and optionally the one or more desirable amino acid (or indicated amino acid) substitutions at the one or more other indicated positions are introduced, is referred to as the “backbone HIV envelope sequence” or “parent HIV envelope sequence.” For example, if positions 302, 304 and 320 in the ConC SOSIP sequence of SEQ ID NO: 3 are mutated to, Met, Vai and Leu respectively, then the ConC SOSIP sequence is considered to be the “backbone” or “parent” sequence. Any HIV envelope protein can be used as the “backbone” or “parent” sequence into which a mutation or substitution according to an embodiment of the invention can be introduced, either alone or in combination with other mutations, such as the so-called SOSIP mutations the mutations in the furin cleavage site and/or the mutations (i)-(vii) of table 1. Non-limiting examples of HIV Env protein that could be used as backbone include HIV Env protein from a natural HIV isolate, a synthetic HIV Env protein, or a consensus HIV Env protein.

According to certain embodiments of the invention, in addition to having Met at position 302, Vai at position 304 and Leu at position 320, the HIV envelope protein can optionally have the indicated amino acid residue at at least one of the indicated positions selected from the group consisting of positions (i)-(xxiii) in Table 1. Typically, it has been seen that HIV Env proteins comprising a combination of at least two, at least three, at least four, at least five, at least six, at least seven, etc of substitutions at the indicated positions (i)- (vii), preferably including further substitution(s) at the indicated positions (viii)-(xxiii), have improved trimerization properties as compared to backbone proteins not having or having less of such substitutions, see e.g. WO 2019/016062. Preferably, at least one of the amino acids in (i)-(xxiii) is introduced into the recombinant HIV Env protein by amino acid substitution. For example, the recombinant HIV Env protein can be produced from an HIV Env protein that contains none, only one or all of the amino acid residues in (i)-(xxiii) above such that all or one or more of the indicated amino acid residues are introduced into the recombinant HIV Env protein by amino acid substitution.

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

In one particular embodiment, the HIV Env backbone protein is a mutant HIV Env protein comprising 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 SOSIP mutant HIV Env protein comprises Cys at positions 501 and 605, and Pro at position 559. According to this embodiment, a recombinant HIV Env protein comprises the amino acid sequence of the SOSIP mutant HIV Env protein and amino acid substitutions at positions 302 resulting in Met in that position, 304 resulting in Vai in that position, 320 resulting in Leu in that position and optionally one or more further amino acid substitutions by the indicated amino acid residue at at least one of the indicated positions selected from the group consisting of entries (i)-(xxii) in Table 1.

The SOSIP mutant HIV Env protein can further comprise a mutation in the furin cleavage site, such as a replacement at positions 608-511 by SEQ ID NO: 6.

In one particular embodiment, the HIV Env backbone protein is an HIV Env consensus clade C comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the HIV consensus clade C sequence of SEQ ID NO: 2 further comprises the so-called SOSIP mutations, i.e., Cys at positions 501 and 605, and Pro at position 559, and in certain embodiments further comprises the so-called SOSIP mutations and a mutation in the furin cleavage site, such as for instance a replacement at positions 508-511 by SEQ ID NO: 6. In a particular embodiment, the HIV Env backbone protein comprises the sequence shown in SEQ ID NO: 3, or a sequence at least 95% identical thereto, wherein amino acids at positions 501, 559, 605, and 508-511 as replaced by SEQ ID NO: 6, are not mutated as compared to SEQ ID NO: 3.

In another particular embodiment, the HIV Env backbone protein is an HIV Env consensus clade B comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the HIV consensus clade B sequence of SEQ ID NO: 4 further comprises the so-called SOSIP mutations, i.e., Cys at positions 501 and 605, and Pro at position 559, and in certain embodiments further comprises the so-called SOSIP mutations and a mutation in the furin cleavage site, such as for instance a replacement at positions 508-511 by SEQ ID NO: 6. In a particular embodiment, the HIV Env backbone protein comprises the sequence shown in SEQ ID NO: 5, or a sequence at least 95% identical thereto, wherein amino acids at positions 501, 559, 605, and 508-511 as replaced by SEQ ID NO: 6, are not mutated as compared to SEQ ID NO: 5.

In yet another particular embodiment, the HIV Env backbone protein is a synthetic HIV Env protein, which may optionally have further SOSIP (501C, 605C, 559P) and/or furin cleavage site mutations (508-51 1 RRRRRR) as described above.

In yet another particular embodiment, the HIV Env backbone protein is a synthetic HIV Env protein, which may optionally have further SOSIP (501C, 605C, 559P) and comprises at least one or preferably at least all of the mutations (i)-(vii) described herein in Table 1. In a preferred embodiment, the HIV Env backbone protein is a synthetic HIV Env protein, which may optionally have further SOSIP (501C, 605C, 559P) and which comprises Pro at position 556, He at position 655, Phe at position 651, Asn at position 535, Vai at position 589, Gin or Glu at position 588, and Vai at position 658.

In yet other particular embodiments, the HIV Env backbone protein is a HIV Env protein from a wild-type clade A, clade B, or clade C HIV virus, optionally comprising additional mutations to repair and/or stabilize the sequence according to methods described in WO 2018/050747 and WO 2019/016062. In a preferred embodiment, the HIV Env backbone protein is a HIV Env protein from a wild-type clade A, clade B, or clade C HIV virus, which may optionally have further SOSIP (501C, 605C, 559P) and which comprises Pro at position 556, He at position 655, Phe at position 651, Asn at position 535, Vai at position 589, Gin or Glu at position 588, and Vai at position 658.

In certain embodiments of the invention, a recombinant HIV Env protein according to the invention (i.e., having Met at position 302, Vai at position 304 and Leu at position 320, and optionally one or more indicated amino acid at positions (i)-(vii) in Table 1 above) can further comprise an indicated amino acid residue (e.g. via substitution) at one or more additional indicated positions selected from the group consisting of positions (vii)-(xxiii) in Table 1. The amino acid substitutions were described previously, e.g. in WO 2019/016062. To the best of the knowledge of the inventors, these previously described mutations were not described in combination with the novel substitutions described herein, i.e. Met at position 302, Vai at position 304 and Leu at position 320. These amino acid mutations in combination with the amino acid substitutions of the invention can further decrease the binding of non- bNAbs and/or stabilize trimer formation. These amino acid substitutions can be introduced into any of the recombinant HIV Env proteins described herein in addition to substitution of Met at position 302, Vai at position 304 and Leu at position 320, and optionally having further substitutions by the indicated amino acid residue at one or more of the indicated positions as described in Table 1. The substitution identified in the present invention [M at position 302, V at position 304 and L at position 320] is to the best of the inventors knowledge not present in natural (group M, i.e. overall) HIV Env sequences, is not found in combination with any of the substitutions (i)-(xxiii) of Table 1 in previously reported HIV Env protein sequences, and was not previously suggested to result in improved trimerization of the HIV Env protein, improved trimer yield and/or increased trimer stability. Clearly, the previously described mutations did not provide any suggestion for introduction of the mutation of the present invention, let alone the surprising effects thereof of decreasing the binding of non-bNAbs. Apart from the point mutations described in Table 1, it is also possible to replace the HR1 loop of the Env protein (amino acid residues 548-568 in a wild- type sequence, with numbering according to gpl60 of the HXB2 isolate) by a shorter and less flexible loop having 7-10 amino acids, preferably a loop of 8 amino acids, e.g. having a sequence chosen from any one of (SEQ ID NOs: 9-14), see e.g. Kong et al (Nat Commun. 2016 Jun 28;7: 12040. doi: 10.1038/ncommsl2040) that describes such shorter loops replacing the HR1 loop. Such an Env variant, further having M at position 302, V at position 304 and L at position 320, and optionally the indicated amino acid residues at at least one of the indicated positions (i)-(vii), is also an embodiment of the invention. Mutations listed in (ix)-(xxiii) can in certain embodiments of the invention be added to HIV Env proteins of the invention, i.e. having M at position 302, V at position 304 and L at position 320. In further embodiments these can be combined with mutations into one or more of the indicated amino acids at positions (i)-(xxiii).

Again, any of those embodiments can be in any HIV Env protein, e.g. a wild-type isolate, a consensus Env, a synthetic Env protein, a SOSIP mutant Env protein, etc. In certain embodiments, the HIV Env protein comprises a sequence that is at least 95% identical to, for example at least 96%, 97%, 98%, 99% identical to, or 100% identical to, any one of SEQ ID NOs: 2-5. For determination of the %identity, preferably positions 302, 304, 320, and preferably in addition the positions (i)-(xxii) of Table 1, and preferably also positions 501, 559 and 605 are not taken into account.

According to embodiments of the invention, a recombinant HIV Env protein decreases binding of non-bNAbs to the clade B and C Env protein compared to an HIV Env protein not having M at position 302, V at position 304 and L at position 320 while further being identical (preferably compared to an HIV Env protein that has N at position 302, R at position 304 and T at position 320 while further being identical).

In another general aspect, the invention provides a nucleic acid molecule encoding a recombinant HIV Env protein according to the invention, and a vector comprising the nucleic acid molecule. The nucleic acid molecules of the invention can be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically. The DNA can be double- stranded or single-stranded. The DNA can for example comprise cDNA, genomic DNA, or combinations thereof. The nucleic acid molecules and vectors can be used for recombinant protein production, expression of the protein in a host cell, or the production of viral particles.

In certain embodiments, the nucleic acid molecules encoding the proteins according to the invention are codon-optimized for expression in mammalian cells, preferably human cells, or insect cells. Methods of codon-optimization are known and have been described previously (e.g. WO 96/09378 for mammalian cells). A sequence is considered codon- optimized if at least one non-preferred codon as compared to a wild-type sequence is replaced by a codon that is more preferred. Herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non- preferred codon. The frequency of codon usage for a specific organism can be found in codon frequency tables, such as in http://www.kazusa.or.jp/codon. Preferably more than one non- preferred codon, preferably most or all non-preferred codons, are replaced by codons that are more preferred. Preferably the most frequently used codons in an organism are used in a codon-optimized sequence. Replacement by preferred codons generally leads to higher expression.

It will be understood by a skilled person that numerous different polynucleotides and nucleic acid molecules can encode the same protein as a result of the degeneracy of the genetic code. It is also understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the protein sequence encoded by the nucleic acid molecules to reflect the codon usage of any particular host organism in which the proteins are to be expressed. Therefore, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may or may not include introns.

Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA and/or RNA synthesis and/or molecular cloning.

Nucleic acid encoding the recombinant HIV Env protein of the invention can for instance also be in the form of RNA, for example mRNA. Such mRNA can be directly used to produce the Env protein, e.g. in cell culture, but also via vaccination, e.g. by administering the mRNA in a drug delivery vehicle such as liposomes or lipid nanoparticles. The nucleic acid or mRNA may also be in the form of self-amplifying RNA or self-replicating RNA, e.g. based on the self-replicating mechanism of positive-sense RNA viruses such as alphaviruses. Such self-replicating RNA (or repRNA or RNA replicon) may be in the form of an RNA molecule expressing alphavirus nonstructural protein genes such that it can direct its own replication amplification in a cell, without producing a progeny virus. For example, a repRNA can comprise 5’ and 3’ alphavirus replication recognition sequences, coding sequences for alphavirus nonstructural proteins, a heterologous gene encoding an antigen, such as the HIV Env protein of the invention, and the means for expressing the antigen, and a polyadenylation tract. Such repRNAs induce transient, high-level antigen expression in a broad range of tissues within a host, and are able to act in both dividing and non-dividing cells. RepRNAs can be delivered to a cell as a DNA molecule, from which a repRNA is launched, packaged in a viral replicon particle (VRP), or as a naked modified or unmodified RNA molecule. In certain embodiments, the mRNA may be nucleoside-modified, e,g, an mRNA or replicating RNA can contain modified nucleobases, such as those described in US2011/0300205. A non-limiting example of repRNA can be found in WO 2019/023566. In non-limiting embodiments, mRNA vaccines and self-amplifying RNA vaccines can for instance include vaccine formats and variations as described in (Pardi et al, 2018, Nature Reviews Drug Discovery 17: 261-279) and in (Zhang et al, 2019, Front. Immunol. 10: 594).

According to certain embodiments of the 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 a promoter. The promoter can be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source). Non-limiting examples of suitable promoters include the human cytomegalovirus immediate early (hCMV IE, or shortly “CMV”) promoter and the Rous Sarcoma virus (RSV) promoter. Preferably, the promoter is located upstream of the nucleic acid within an expression cassette.

The nucleic acid according to the invention may be incorporated into a vector. In certain embodiments a vector comprises DNA and/or RNA. According to embodiments of the invention, a vector can be an expression vector. Expression vectors include, but are not limited to, vectors for recombinant protein expression and vectors for delivery of nucleic acid into a subject for expression in a tissue of the subject, such as a viral vector. Examples of viral vectors suitable for use with the invention include, but are not limited to adenoviral vectors, adeno-associated virus vectors, pox virus vectors, Modified Vaccinia Ankara (MV A) vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, alphavirus vectors, etc. The vector can 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, etc. In certain embodiments of the invention, the vector is an adenovirus vector, e.g., a recombinant adenovirus vector. A recombinant adenovirus vector may for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd). Preferably, an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc. In other embodiments, an adenovirus vector is a rhAd vector, e.g. rhAd51, rhAd52 or rhAd53. In other embodiments, the recombinant adenovirus is based upon a chimpanzee adenovirus such as ChAdOx 1 (see e.g. WO 2012/172277), or ChAdOx 2 (see e.g. WO 2018/215766), or BZ28 (see e.g. WO 2019/086466). In other embodiments, the recombinant adenovirus is based upon a gorilla adenovirus such as BLY6 (see e.g. WO 2019/086456), or BZl (see e.g. WO 2019/086466).

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

According to embodiments of the invention, any of the recombinant HIV Env proteins described herein can be expressed and/or encoded by any of the vectors described herein. In view of the degeneracy of the genetic code, the skilled person is well aware that several nucleic acid sequences can be designed that encode the same protein, according to methods entirely routine in the art. The nucleic acid encoding the recombinant HIV Env protein of the invention can optionally be codon-optimized to ensure proper expression in the host cell (e.g., bacterial or mammalian cells). Codon-optimization is a technology widely applied in the art.

The invention also provides cells, preferably isolated cells, ex vivo cells or in vitro cells, comprising any of the nucleic acid molecules and vectors described herein. The cells can for instance be used for recombinant protein production, or for the production of viral particles.

Embodiments of the invention thus also relate to a method of making a recombinant HIV Env protein. The method comprises transfecting a host cell with an expression vector comprising nucleic acid encoding a recombinant HIV Env protein according to an embodiment of the invention operably linked to a promoter, growing the transfected cell under conditions suitable for expression of the recombinant HIV Env protein, and optionally purifying or isolating the recombinant HIV Env protein expressed in the cell. The recombinant HIV Env protein can be isolated or collected from the cell by any method known in the art including affinity chromatography, size exclusion chromatography, etc. Techniques used for recombinant protein expression will be well known to one of ordinary skill in the art in view of the present disclosure. The expressed recombinant HIV Env protein can also be studied without purifying or isolating the expressed protein, e.g., 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 permit association of the protein so as to form the trimeric complex. For example, mammalian cells transfected with an expression vector encoding the recombinant HIV Env protein operably linked to a promoter (e.g. CMV promoter) can be cultured at 33-39°C, e.g. 37°C, and 2-12% CO2, e.g. 8% CO2. Expression can also be performed in alternative expression systems such as insect cells or yeast cells, all conventional in the art. The expressed HIV Env protein can then be isolated from the cell culture for instance by lectin affinity chromatography, which binds glycoproteins. The HIV Env protein bound to the column can be eluted with mannopyranoside. The HIV Env protein eluted from the column can be subjected to further purification steps, such as size exclusion chromatography, as needed, to remove any residual contaminants, e.g., cellular contaminants, but also Env aggregates, gpl40 monomers and gpl20 monomers. Alternative purification methods, non-limiting examples including antibody affinity chromatography, negative selection with non-bNAbs, anti-tag purification, or other chromatography methods such as ion exchange chromatography etc, as well as other methods known in the art, could also be used to isolate the expressed HIV Env protein.

The nucleic acid molecules and expression vectors encoding the recombinant HIV Env proteins of the invention can be made by any method known in the art in view of the present disclosure. For example, nucleic acid encoding the recombinant HIV Env protein can be prepared by introducing mutations that encode the one or more amino acid substitutions at the indicated positions into the backbone HIV envelope sequence using genetic engineering technology and molecular biology techniques, e.g., site directed mutagenesis, polymerase chain reaction (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 a host cell, and the expressed protein purified from the cell culture by any method known in the art in view of the present disclosure.

In another general aspect, the invention relates to a trimeric complex comprising a noncovalent oligomer of three of the recombinant HIV Env proteins according to the invention. The trimeric complex can comprise any of the recombinant HIV Env proteins described herein. Preferably the trimeric complex comprises three identical monomers (or identical heterodimers if gpl40 is cleaved) of the recombinant HIV Env proteins according to the invention. The trimeric complex can be separated from other forms of the HIV envelope protein, such as the monomer form, or the trimeric complex can be present together with other forms of the HIV envelope protein, such as the monomer form.

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

A carrier can include one or more pharmaceutically acceptable excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings. The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes. For liquid injectable preparations, for example, suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like. For solid oral preparations, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. For nasal sprays/inhalant mixtures, the aqueous solution/suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives.

Compositions of the invention can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections. The parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection. Compositions of the invention can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.

Embodiments of the invention also relate to methods of making the composition. According to embodiments of the invention, a method of producing a composition comprises mixing a recombinant HIV Env protein, trimeric complex, isolated nucleic acid, vector, or host cell of the invention with one or more pharmaceutically acceptable carriers. One of ordinary skill in the art will be familiar with 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 the HIV antigens have previously been used in immunogenic compositions and vaccines for vaccinating a subject against an HIV infection, or for generating an immune response against an HIV infection in a subject. As used herein, “subject” means any animal, preferably a mammal, most preferably a human, to who will be or has been administered an immunogenic composition according to embodiments of the invention. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., preferably a human. The recombinant HIV Env proteins of the invention can also be used as antigens to induce an immune response against human immunodeficiency virus (HIV) in a subject in need thereof. The immune response can be against one or more HIV clades, such as clade A, clade B, clade C, etc. The compositions can comprise a vector from which the recombinant HIV Env protein is expressed, or the composition can comprise an isolated recombinant HIV Env protein according to an embodiment of the invention.

For example, compositions comprising a recombinant HIV protein or a trimeric complex thereof can be administered to a subject in need thereof to induce an immune response against an HIV infection in the subject. A composition comprising a vector, such as an adenovirus vector, encoding a recombinant HIV Env protein of the 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 against an HIV infection in the subject. The methods described herein also include administering a composition of the 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 adenovirus vectors or MV A vectors, including methods of priming and boosting an immune response. In certain embodiments, the HIV Env protein can be displayed on a particle, such as a liposome, virus-like particle (VLP), nanoparticle, virosome, or exosome, optionally in combination with endogenous and/or exogenous adjuvants. When compared to soluble or monomeric Env protein on its own, such particles typically display enhanced efficacy of antigen presentation in vivo.

Examples of VLPs that display HIV Env protein can be prepared e.g. by co- expressing the HIV Env protein with self-assembling viral proteins such as HIV Gag core or other retroviral Gag proteins. VLPs resemble viruses, but are non-infectious because they contain no viral genetic material. The expression of viral structural proteins, such as envelope or capsid, can result in self-assembly of VLPs. VLPs are well known to the skilled person, and their use in vaccines is for instance described 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. The HIV Env trimer proteins can for instance be non- covalently coupled to such liposomes by electrostatic interactions, e.g. by adding a His-tag to the C-terminus of the HIV Env trimer and a bivalent chelating atom such as Ni2+ or Co2+ incorporated into the head group of derivatized lipids in the liposome. In certain non-limiting and exemplary embodiments, the liposome comprises 1,2-di stearoyl -sn-glycero-3- phosphocholine (DSPC), cholesterol, and the Nickel or Cobalt salt of 1,2-dioleoyl-sn- glycero-3-[(N-(5 -amino- l-carboxypentyl)iminodiacetic acid)succinyl] (DGS-NTA(Ni2+) or DGS-NTA(Co2+)) at 60:36:4 molar ratio. In preferred embodiments, the HIV Env trimer proteins are covalently coupled to the liposomal surface, e.g. via a maleimide functional group integrated in the liposome surface. In certain non-limiting exemplary embodiments thereof, the liposome comprises DSPC, cholesterol, and l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] lipid in a molar ratio of 54:30: 16. The HIV Env protein can be coupled thereto e.g. via an added C-terminal cysteine in the HIV Env protein. The covalently coupled variants are more stable, elicit high antigen specific IgG titers and epitopes at the antigenically less relevant ‘bottom’ of the Env trimer are masked. Methods for preparing HIV Env trimers coupled to liposomes, as well as their characterization, are known and have for instance been described in (Bale et al, 2017), incorporated by reference herein. The invention also provides an HIV Env protein of the invention fused to and/or displayed on a liposome.

In certain embodiments, a HIV Env protein of the invention is fused to self- assembling particles, or displayed on nanoparticles. Antigen nanoparticles are assemblies of polypeptides that present multiple copies of antigens, e.g. the HIV Env protein of the instant invention, which result in multiple binding sites (avidity) and can provide improved antigen stability and immunogenicity. Preparation and use of self-assembling protein nanoparticles for use in vaccines is well-known to the skilled person, see e.g. (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 nanoparticles displaying proteins on their surface are for instance described in WO2011/082087. Description of trimeric HIV-1 antigens on such particles has for instance been described in (He et al, 2016). Other self-assembling protein nanoparticles as well as preparation thereof, are for instance disclosed in WO 2014/124301, and US 2016/0122392, incorporated by reference herein. The invention also provides an HIV Env protein of the invention fused to and/or displayed on a self-assembling nanoparticle. The invention also provides compositions comprising VLPs, liposomes, or self- assembling nanoparticles according to the invention.

In certain embodiments, an adjuvant is included in a composition of the invention or co-administered with a composition of the invention. Use of adjuvant is optional, and may further enhance immune responses when the composition is used for vaccination purposes. Adjuvants suitable for co-administration or inclusion in compositions in accordance with the invention should preferably be ones that are potentially safe, well tolerated and effective in people. Such adjuvants are well known to the skilled person, 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, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Aluminium salts such as Aluminium Phosphate (e.g. AdjuPhos) or Aluminium Hydroxide, and MF59.

Also disclosed herein are recombinant HIV envelope proteins comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, which represent the HIV envelope consensus clade C and consensus clade B sequences, respectively A recombinant HIV envelope protein comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 can optionally further comprise the so-called SOSIP mutations and/or a mutation in the furin cleavage site, such as, for instance in those sequences shown in SEQ ID NO: 3, or SEQ ID NO: 3 further comprising 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 determining the %identity for these sequences, the amino acids at the mutated furin cleavage site and at positions 501, 605, 559, 556 and 558 are preferably not taken into account. Such proteins are expressed at high levels and have a high level of stability and trimer formation. Such HIV Env proteins can in certain embodiments be used as backbone proteins, wherein the mutation of R304 into V, N302 into M and T320 into L can be made to obtain a molecule of the invention. Isolated nucleic acid molecules encoding these sequences, vectors comprising these sequences operably linked to a promoter, and compositions comprising the protein, isolated nucleic acid molecule, or vector are also disclosed.

In another aspect, the invention provides for a method of improving the trimer folding of an HIV Env protein to obtain higher quality trimers with reduced non-bNAb binding, the method comprising introducing the substitution of: Asn at position 302 by Met, Arg at position 304 by Vai, and Thr at position 320 by Leu into a parent HIV Env protein, wherein the numbering of the positions is according to the numbering in gpl60 of HIV-1 isolate HXB2.

EXAMPLES

The following examples of the invention are to further illustrate the nature of the invention. It should be understood that the following examples do not limit the invention and that the scope of the invention is to be determined by the appended claims.

Presentation of misfolded Env trimers on the surface of HIV virions is thought to be one of the many ways HIV is able to evade the immune system. These misfolded Env expose immunodominant sites and elicit non-neutralizing antibodies (non-bNAbs), while these non- bNAbs may bind to the virus particles, they do not neutralize viral infectivity and are therefore undesired. It has been observed that introducing Met at position 302, Vai at position 304 and Leu at position 320 into the sequence of an HIV envelope protein stabilizes the apex in a closed conformation, which results in a decrease in non-bNAb binding.

Example 1: Mutation of HIV Envelope at position 302 into Met, at position 304 into Vai and at position 320 into Leu decreases the binding of non-bNAbs

Materials and methods

Analytical SEC

The HIV Env variants were expressed in 96 well format cell cultures. An ultra high- performance liquid chromatography system (Vanquish, Thermo Scientific) and pDAWN TREOS instrument (Wyatt) coupled to an Optilab pT-rEX Refractive Index Detector (Wyatt) in combination with an in-line Nanostar DLS reader (Wyatt) was used for performing the analytical SEC experiment. The cleared crude cell culture supernatants were applied to a TSK-Gel UP-SW3000 4.6x150 mm column with the corresponding guard column (Tosoh Bioscience) equilibrated in running buffer (150 mM sodium phosphate, 50 mM sodium chloride, pH 7.0) at 0.3 mL/min. When analyzing supernatant samples, pMALS detectors were offline and analytical SEC data was analyzed using Chromeleon 7.2.8.0 software package. The signal of supernatants of non-transfected cells was subtracted from the signal of supernatants of HIV Env transfected cells.

AlphaLISA®

AlphaLISA® (Perkin-Elmer) is a bead-based proximity assay in which singlet oxygen molecules generated by high energy irradiation of Donor beads transfers to Acceptor beads which are within a distance of approximately 200 nm. It is a sensitive high throughput screening assay that does not require washing steps. A cascading series of chemical reactions results in a chemiluminescent signal (Eglen et al. Curr Chem Genomics, 2008). For the AlphaLISA® assay the constructs were equipped with a sortase A-Flag-His tag (AAALPETGGGSDYKDDDDKP(GGGGS)7H6 (SEQ ID NO: 15)). The HIV constructs were expressed in Expi293F cells, which were cultured for 3 days in 96 well plates (200 pl/well). Crude supernatants were diluted 120 times in AlphaLISA® buffer (PBS + 0.05% Tween-20 + 0.5 mg/mL BSA) except for 17b-based assays, in which supernatants were diluted 12 times. Subsequently 10 pl of these dilutions were transferred to a half-area 96-well plate and mixed with a 40pl mix of acceptor beads, donor beads and mAb. The beads were mixed well before use. After 2 hours of incubation at RT, non-shaking, the signal was measured with Neo (BioTek) The donor beads were conjugated to ProtA (Cat#: AS102M, Perkin Elmer), which could bind to the mAb. The acceptor beads were conjugated to an anti- His antibody (Cat#: AL112R, Perkin Elmer) to detect the His-tag of the protein. For the quantification of the total protein level, a combination of Nickel -conjugated donor beads (Cat#: AS 10 IM, Perkin Elmer) together with acceptor beads carrying anti -Flag antibody (Cat#: AL112R, Perkin Elmer) were used. For 17b in combination with sCD4-His, a combination of ProtA donor beads and anti -Flag acceptor beads was used. The average signal of mock transfections (no Env) was subtracted from the AlphaLISA counts measured for the different Env proteins. As a reference the ConC SOSIP Env plasmid was used.

The monoclonal antibodies (mAbs) that were used for analysis are well known in the field (see e.g. WO 2018/050747), and are indicated in Table 2 with some of their features. Table 2: HIV Env antibodies used in experiments

Flow cytometry

HEK293 cells (0.4* 10⁶ cells/well) were seeded in 6-well plates and after overnight growth transfected with 1 ug HIV Env and 0.3 ug GFP DNA for 48 hrs. Cells were detached, washed with PBS and stained with LIVE/DEADTM Fixable Violet Dead Cell Stain Kit (Invitrogen). For surface staining, cells were washed and incubated with anti-gpl20 HIV antibodies (2G12, PGT128, PGT145, VRC26, F105, 14E, 17b) for 30 min at 4°C in FACS buffer (PBS with 0.5% BSA). Cells were washed and stained with goat anti-Human IgG Alexa Fluor 647 (Invitrogen) secondary antibody for 30 min at 4°C in FACS buffer. Cells were washed and fixed with lx BD CellFIX (BD Biosciences) for 15 min. Cells were washed and resuspended in FACS buffer before analysis on a FACS Canto instrument (BD Biosciences). Data were analyzed with FlowJoTM Software (Becton, Dickinson and Company) and plotted as median fluorescence intensity of the single, live cell population.

Results

In ConB SOSIP the two mutations N302M and T320L decrease the binding of non-bNAbs, whereas the binding of bNAb PGT128 increased, indicating an increased quality of Env. The same is true for the R304V substitution, which reduces non-bNAb binding but increases PGT145 binding. When combining N302N and T320L with R304V a greater reduction in the V3 loop binding Abs (447-52D and 14E) is seen than expected, indicating a synergistic effect (Fig. 2). The effect of N302M, T320L and R304V on quality of ConB SOSIP is also seen in SEC-MALS where both the combination of N302M and T320L, but also R304V increase trimer yield and decrease monomer yield (Fig. 2) Even in the presence of additional strong stabilizing substitutions (L556P, K655I, N651F, I535N, D589V, K588E, Q658V) the effect of the three stabilizing mutations (N302M, T320L and R304V) still increases expression levels of the gpl40 trimer (Fig. 3). Since R304 is directly adjacent to the 447-52D epitope and it is not known whether it is in the epitope of 14E, we tested whether we restore binding to 447-52D and 14E, which bind to a linear epitope, by denaturation of the Env protein with the three mutations. By incubation of the ConB_SOSIP_N302M_T320L_R304V at 80°C for 5 minutes, the binding of the non-bNAbs to linear epitopes is somewhat restored (Fig. 4), indicating that the three mutations do not directly modify the epitopes, but that the reduced binding is likely due to concealing the non-bNAb epitopes by closing the Env protein. N302M and T320L, and also R304V decrease the binding of non-bNAbs in two soluble clade C Envs (Figs 5 and 6). The stability increase is so strong that the stabilizing covalent bonds of the disulfides SOS (between cysteine 501 and 605) and DS (between cysteine 201 and 433) are no longer needed. Moreover, the three substitutions also show a reduction of non-bNAbs binding when they are added in the full length, membrane-bound ConC, indicating the stabilizing substitutions can also be applied to full length Envs (Fig. 7-9).

SEQ ID NO: 6 (furin cleavage site mutant sequence)

RRRRRR

SEQ ID NO: 7 (example of a signal sequence (e.g. used for ConC SOSIP)) MRVRGILRNWQQWWIWGILGFWMLMICNWG (note: the last VG could be the beginning of the mature protein or the end of the signal sequence)

SEQ ID NO: 8 (example of a signal sequence (e.g. used for ConB SOSIP)

MRVKGI RKNYQHLWRWGTMLLGMLMI CSA

SEQ ID NO: 9 (example of 8 amino acid sequence that can replace HR1 loop) NPDWLPDM

SEQ ID NO: 10 (example of 8 amino acid sequence that can replace HR1 loop) GSGSGSGS

SEQ ID NO: 11 (example of 8 amino acid sequence that can replace HR1 loop) DDVHPDWD

SEQ ID NO: 12 (example of 8 amino acid sequence that can replace HR1 loop) RDTFALMM

SEQ ID NO: 13 (example of 8 amino acid sequence that can replace HR1 loop) DEEKVMDF

SEQ ID NO: 14 (example of 8 amino acid sequence that can replace HR1 loop) DEDPHWDP

Claims

1. A recombinant human immunodeficiency virus (HIV) envelope (Env) protein, comprising the following amino acid residues: Met at position 302, Vai at position 304, and Leu at position 320, wherein the numbering of the positions is according to the numbering in gpl60 of HIV-1 isolate HXB2.

2. The recombinant HIV Env protein according to claim 1, further comprising at least one of the following:

(a) Cys at positions 501 and 605;

(b) Pro at position 559; or

(c) Cys at positions 501 and 605 and Pro at position 559.

3. The recombinant HIV Env protein according to claim 1 or 2, further comprising one or more of the following amino acid residues at the indicated positions:

(i) Pro at position 556

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

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

(iv) Asn or Gin, preferably Asn, at position 535;

(v) Vai, He or Ala, preferably Vai, at position 589;

(vi) Gin, Glu, He, Met, Vai, Trp, or Phe, preferably Gin or Glu, at position 588;

(vii) Vai, He, Phe, Met, Ala, and Leu, preferably Vai at position 658.

(viii) 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;

(ix) Pro at position 329;

(x) Asp at position 586;

(xi) He at position 204;

(xii) Phe or Trp, preferably Phe, at position 573

(xiii) Lys at position 64 or Arg at position 66 or Lys at position 64 and Arg at position 66;

(xiv) Trp at position 316;

(xv) Pro at position 558 or at both positions 556 and 558; (xvi) replacement of the loop at amino acid positions 548-568 (HRl-loop) by a loop having 7-10 amino acids, preferably a loop of 8 amino acids, for example having a sequence chosen from any one of (SEQ ID NOs: 9-14);

(xvii) or Arg at position 519, or Arg at position 520, or Arg at both positions 519 and 520;

(xviii) His at position 108;

(xix) His at position 538,

(xx) Trp, Phe, Met, or Leu, preferably Trp at position 650;

(xxi) a mutation in a furin cleavage sequence of the HIV Env protein, preferably a replacement at positions 508-511 by RRRR.R.R. (SEQ ID NO: 6); and/or

(xxii) Cys at both positions 201 and 433.

4. The recombinant HIV Env protein according any one of claims 1-3, comprising Pro at position 556, He at position 655, Phe at position 651, Asn at position 535, Vai at position 589, Glu at position 588, and Vai at position 658.

5. The recombinant HIV Env protein according to any one of the preceding claims, wherein the HIV Env protein is a clade A, B, or C HIV Env protein.

6. The recombinant HIV Env protein according to any one of the preceding claims, being a gpl40 or gpl60 protein, or an Env protein having a truncation in the cytoplasmic region.

7. A trimeric complex comprising a noncovalent oligomer of three identical recombinant HIV Env proteins of any one of claims 1-6.

8. A particle, preferably a liposome or nanoparticle, displaying on its surface a recombinant HIV Env protein according to any one of claims 1-6 or a trimeric complex of claim 7.

9. An isolated nucleic acid molecule encoding a recombinant HIV Env protein of any one of claims 1-6.

10. A vector comprising the isolated nucleic acid molecule of claim 9 operably linked to a promoter.

11. The vector of claim 10, which is an adenovirus vector.

12. A host cell comprising the isolated nucleic acid molecule of claim 9 or the vector of claim 10 or 11.

13. A method of producing a recombinant HIV Env protein, comprising growing the host cell of claim 12.

14. A composition comprising the recombinant HIV Env protein according to any of claims 1-6, the trimeric complex of claim 7, the particle of claim 8, the isolated nucleic acid molecule of claim 9, or the vector of claim 10 or 11, and a pharmaceutically acceptable carrier.

15. A method of improving the trimer folding of an HIV Env protein, the method comprising introducing the substitution of:

- the amino acid in position 302 by Met;

- the amino acid in position 304 by Vai; and

- the amino acid in position 320 by Leu, into a parent HIV Env protein, wherein the numbering of the positions is according to the numbering in gpl60 of HIV-1 isolate HXB2.