Patent Application
20220380414
The present invention generally relates to Human Immunodeficiency Virus (HIV) infection, and more specifically to compositions and methods for research and therapeutic applications relating to HIV infection.
The human immunodeficiency virus type 1 (HIV-1) continues to infect more than 2.1 million individuals annually for an estimated total of 37 million people living with this virus in 2016. Enormous efforts have been made to improve the clinical management of HIV/AIDS through highly-active antiretroviral drugs (HAART). Accordingly, HIV infection can be controlled with HAART which, in most cases, allows for a significant increase in the life expectancy of infected individuals. Unfortunately, HAART is unable to fully restore health or a normal immune status. HAART-treated individuals still experience several co-morbidities including increased cardiovascular disease, bone disorders and cognitive impairment. Most importantly, due to the presence of latent viral reservoirs, persisting mainly in long-lived memory CD4+ T cells, therapy interruption leads to the re-emergence of viral replication and AIDS progression. Therefore, the development of new approaches aimed at eradicating or functionally curing HIV infection are desperately needed.
HIV-1 entry is mediated by the interaction of HIV-1 envelope glycoproteins (Env) with the CD4 receptor and either CCR5 or CXCR4 chemokine coreceptors on T cells. Env is exposed on the surface of viral particles and infected cells as three gp120 exterior glycoproteins non-covalently associated with three gp41 transmembrane glycoproteins (gp120-gp41)3(1-3). Binding of gp120 to the CD4 receptor leads to major conformational changes in gp120, resulting in the rearrangement of the V1, V2 and V3 loops, and the formation of the coreceptor binding site (CoRBS) and the bridging sheet (4-11). CD4 interaction also leads to the exposure of a gp41 helical heptad repeat (HR1) (12). Subsequent interaction of gp120 with the coreceptor triggers additional conformational changes in gp41, resulting in the formation of a six-helix bundle formed by HR1 and HR2 heptad repeats and the fusion of viral and cellular membranes (12-14).
Recent studies have shown that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered “closed” conformation (state 1) through a default intermediate conformation (state 2) to an “open” conformation in which it is bound to three CD4 receptor molecules (state 3) (Munro, J. B. et al., Science 346, 759-763 (2014); Herschhorn, A. et al., mBio 7, e01598-e16 (2016); Ma, X. et al., eLife 7, e34271 (2018)). The pre-triggered state 1 conformation of viral Env is preferentially stabilized by many broadly neutralizing antibodies, and thus of interest for the design of immunogens, whereas the state 3 conformation is of interest for the development of small CD4 mimetics or additional type of small inhibitors with the capacity to stabilize Env in more “open” conformations such as states 2, 2A and 3. However, there is currently no approach to induce the viral Env to adopt a given conformation, which would be useful for studying the Env structure as well as for the development of HIV vaccines and entry inhibitors.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
The present disclosure provides the following items 1 to 32:
1. A composition comprising:
(i) a mutated HIV-1 gp120 polypeptide from an HIV-1 strain, wherein the native residues at positions 61, 105, 108, 375, 474, 475 and 476 in the HIV-1 strain are substituted, and wherein
(ii) a gp120 or gp41 ligand.
2. The composition of item 1, wherein the HIV-1 strain is a CRF01_AE strain and the mutated HIV-1 gp120 polypeptide comprises one or more of the following substitutions: H61Y, Q105H, V1081, H375T or H375S, N474D, 1475M, and K476R.
3. The composition of item 2, wherein the mutated HIV-1 gp120 polypeptide comprises the following substitutions: (1) H61Y, (2) Q105H, (3) V1081, (4) H375T, (5) N474D, (6) 1475M, and (7) K476R.
4. The composition of item 2, wherein the mutated HIV-1 gp120 polypeptide comprises the following substitutions: (1) H61Y, (2) Q105H, (3) V1081, (4) H375S, (5) N474D, (6) 1475M, and (7) K476R.
5. The composition of item 1, wherein the HIV-1 strain is a clade A, B, C, D, G or H HIV-1 strain, and the mutated HIV-1 gp120 polypeptide comprises the following substitutions: Y61H, H105Q, 1108V, S375H, D474N, M4751, and R476K.
6. The composition of item 1, wherein the HIV-1 strain is a clade F HIV-1 strain, and the mutated HIV-1 gp120 polypeptide comprises the following substitutions: Y61H, H105Q, 1108V, S375H, N474D, M4751, and K476R.
7. The composition of item 1, wherein the HIV-1 strain is a clade J HIV-1 strain, and the mutated HIV-1 gp120 polypeptide comprises the following substitutions: Y61H, H105Q, 1108V, S375H, D474N, M4751, and K476R.
8. The composition of item 1, wherein the HIV-1 strain is a clade K HIV-1 strain, and the mutated HIV-1 gp120 polypeptide comprises the following substitutions: Y61H, H105Q, 1108V, 1375H, D474N, M4751, and R476K.
9. The composition of any one of items 1 to 8, wherein the mutated HIV-1 gp120 polypeptide is an HIV envelope trimer.
10. The composition of any one of items 1 to 9, wherein the gp120 ligand induces said Env trimer into an open state 2/3 conformation.
11. The composition of item 10, wherein the gp120 ligand is a CD4 mimetic (CD4mc).
12. The composition of item 11, wherein said CD4mc is the following compound:
13. The composition of any one of items 1 to 9, wherein the gp120 ligand induces said Env trimer into a closed state 1 conformation.
14. The composition of item 13, wherein the gp120 ligand is a conformational blocker.
15. The composition of item 13 or 14, wherein the gp120 ligand is one of the following compounds:
16. The composition of any one of items 1 to 15, further comprising a vaccine adjuvant.
17. The composition of any one of items 1 to 16, wherein said mutated HIV-1 gp120 polypeptide is comprised in a cell, a liposome or a virus-like particle (VLP).
18. A method for eliciting an immune response to HIV-1 in a subject, comprising administering to the subject a prophylactically or therapeutically effective amount of (i) the mutated HIV-1 gp120 polypeptide defined in any one of items 1 to 9 and 17, and (ii) a gp120 ligand.
19. The method of item 18, comprising administering to the subject a prophylactically or therapeutically effective amount of the composition of any one of items 1 to 17.
20. The method of item 18 or 19, wherein said subject is not infected by HIV-1.
21. The method of item 18 or 19, wherein said subject is infected by HIV-1.
22. A method for determining whether a test agent binds to an HIV Env trimer into an open state 2/3 conformation comprising contacting said test agent with the mutated HIV-1 gp120 polypeptide defined in any one of items 1 to 9 and 17, and the gp120 ligand defined in any one of items 10 to 12.
23. A method for determining whether a test agent binds to an HIV Env trimer into a closed state 1 conformation comprising contacting said test agent with the mutated HIV-1 gp120 polypeptide defined in any one of items 1 to 9 and 17, and the gp120 ligand defined in any one of items 13 to 15.
24. A method for inducing an HIV Env trimer into an open state 2/3 conformation comprising contacting an HIV Env trimer comprising the mutated HIV-1 gp120 polypeptide defined in any one of items 1 to 9 and 17 with the gp120 ligand defined in any one of items 10 to 12.
25. A method for inducing an HIV Env trimer into an open state 1 conformation comprising contacting an HIV Env trimer comprising the mutated HIV-1 gp120 polypeptide defined in any one of items 1 to 9 and 17 with the gp120 ligand defined in any one of items 13 to 15.
26. The method of item 22 or 24, comprising the mutated HIV-1 gp120 polypeptide defined in item 3.
27. The method of item 23 or 25, comprising the mutated HIV-1 gp120 polypeptide defined in item 4.
28. A method for determining whether a test agent induces a closed (state 1) conformation of an HIV Env trimer comprising (a) contacting the mutated HIV-1 gp120 polypeptide defined in any one of items 1 to 9 and 17 with said test agent, and (b) determining whether the HIV Env trimer is in a closed (state 1) conformation.
29. A method for determining whether a test agent induces an open (state 2/3) conformation of an HIV Env trimer comprising (a) contacting the mutated HIV-1 gp120 polypeptide defined in any one of items 1 to 9 and 17 with said test agent, and (b) determining whether the HIV Env trimer is in an open (state 2/3) conformation.
30. Use of (i) the mutated HIV-1 gp120 polypeptide defined in any one of items 1 to 9 and 13, and (ii) a gp120 ligand, for eliciting an immune response to HIV-1 in a subject.
31. Use of (i) the mutated HIV-1 gp120 polypeptide defined in any one of items 1 to 9 and 13, and (ii) a gp120 ligand, for the manufacture of a medicament for eliciting an immune response to HIV-1 in a subject.
32. A complex or composition comprising (i) the mutated HIV-1 gp120 polypeptide defined in any one of items 1 to 9 and 13, and (ii) a gp120 ligand, for use in eliciting an immune response to HIV-1 in a subject.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
In the studies described herein, the present inventors have shown that introducing certain mutations in the HIV Env protein “re-shapes” the Phe43 cavity and makes the HIV env protein amenable to adopt specific conformations when contacted with gp120 ligands. More specifically, full-length gp160 glycoprotein constructs from CRF01_AE strains comprising the six mutations H61Y, Q105H, V1081, N474D, 1475M, and K476R, combined with H375T, tend to adopt an open state 2/3 configuration in the presence of the CD4 mimetic BNM-III-170, whereas full-length gp160 glycoprotein constructs from CRF01_AE strains comprising the same six mutations, combined with H375S, tend to adopt a state 1 configuration in the presence of the HIV-1 attachment inhibitor Temsavir (BMS-626529), a conformational blocker.
Accordingly, in a first aspect, the present disclosure provides a complex or composition comprising:
(i) a mutated HIV-1 gp120 polypeptide from an HIV-1 strain, wherein the native residues at positions 61, 105, 108, 375, 474, 475 and 476 in the HIV-1 strain (numbering based on the amino acid sequence of Envelope glycoprotein gp160 from the HXBc2 strain of HIV-1, UniProtKB accession No. P04578.2, SEQ ID NO:6) are substituted, and wherein (a) the HIV-1 strain is a CRF01_AE strain and the native residues at positions 61, 105, 108, 375, 474, 475 and 476 are H, Q, V, H, N, I and K, respectively; (b) the HIV-1 strain is a Clade A, B, C, D, G or H strain, and the native residues at positions 61, 105, 108, 375, 474, 475 and 476 are Y, H, I, S, D, M and R, respectively; (c) the HIV-1 strain is a Clade F strain, and the native residues at positions 61, 105, 108, 375, 474, 475 and 476 are Y, H, I, S, N, M and K, respectively; (d) the HIV-1 strain is a Clade J strain, and the native residues at positions 61, 105, 108, 375, 474, 475 and 476 are Y, H, I, S, D, M and K, respectively; or (e) the HIV-1 strain is a Clade K strain, and the native residues at positions 61, 105, 108, 375, 474, 475 and 476 are Y, H, I, I, D, M and R, respectively; and
(ii) a gp120 or gp41 ligand.
In another aspect, the present disclosure provides a method for rendering an HIV Env trimer from an HIV strain more amenable to adopt a closed or open conformation following binding of a gp120 or gp41 ligand, comprising introducing amino acid substitutions at positions 61, 105, 108, 375, 474, 475 and 476 in the gp120 protein forming said HIV Env trimer.
The numbering used in the disclosed HIV-1 Env proteins is relative to the HXBc2 strain of HIV-1 (UniProtKB/Swiss-Prot: P04578.2,
As used herein, the term “clade” refers to related human immunodeficiency viruses (HIVs) classified according to their degree of genetic similarity. A clade generally refers to a distinctive branch in a phylogenetic tree. There are currently four major groups of HIV-1 isolates: M, N, O and P. Group M (the Main group) is responsible for the majority of cases in the global pandemic and consists of 9 major clade subtypes (A1, A2, B, C, D, F1, F2, G, H, J, and K) and many circulating recombinant forms (CRFs).
In an embodiment, the substitution is with an amino acid that is present at high frequency (e.g., more than 30%, 40% or 50%, preferably more than 60%) at the corresponding position in another HIV strain or clade.
In an embodiment, the mutated HIV-1 gp120 polypeptide is from a circulating recombinant form (CRF), more specifically CRF01_AE. As shown in
In another embodiment, the mutated HIV-1 gp120 polypeptide is from a clade A, B, C, D, G or H HIV-1 strain, and the mutated HIV-1 gp120 polypeptide comprises the following substitutions: Y61H, H105Q, 1108V, S375H, D474N, M4751, and R476K.
In another embodiment, the mutated HIV-1 gp120 polypeptide is from a clade F HIV-1 strain, and the mutated HIV-1 gp120 polypeptide comprises the following substitutions: Y61H, H105Q, 1108V, S375H, N474D, M4751, and K476R.
In another embodiment, the mutated HIV-1 gp120 polypeptide is from a clade J HIV-1 strain, and the mutated HIV-1 gp120 polypeptide comprises the following substitutions: Y61H, H105Q, 1108V, S375H, D474N, M4751, and K476R.
In another embodiment, the mutated HIV-1 gp120 polypeptide is from a clade K HIV-1 strain, and the mutated HIV-1 gp120 polypeptide comprises the following substitutions: Y61H, H105Q, 1108V, 1375H, D474N, M4751, and R476K.
In another embodiment, the mutated HIV-1 gp120 polypeptide is from a clade A and comprises an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with residues 33-493 of SEQ ID NO: 7 (clade A1) or residues 32-491 of SEQ ID NO: 8 (clade A2).
In another embodiment, the mutated HIV-1 gp120 polypeptide is from a clade B and comprises an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with residues 33-496 of SEQ ID NO: 9.
In another embodiment, the mutated HIV-1 gp120 polypeptide is from a clade C and comprises an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with residues 33-483 of SEQ ID NO: 10.
In another embodiment, the mutated HIV-1 gp120 polypeptide is from a clade D and comprises an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with residues 33-495 of SEQ ID NO: 11.
In another embodiment, the mutated HIV-1 gp120 polypeptide is from a clade F and comprises an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with residues 33-487 of SEQ ID NO: 12 (clade F1) or 33-486 of SEQ ID NO: 13 (clade F2).
In another embodiment, the mutated HIV-1 gp120 polypeptide is from a clade G and comprises an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with residues 33-490 of SEQ ID NO: 14.
In another embodiment, the mutated HIV-1 gp120 polypeptide is from a clade H and comprises an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with residues 33-494 of SEQ ID NO: 15.
“Identity” refers to sequence identity between two polypeptides. Identity can be determined by comparing each position in the aligned sequences. Methods of determining percent identity are known in the art, and several tools and programs are available to align amino acid sequences and determine a percentage of identity including EMBOSS Needle, ClustalW, SIM, DIALIGN, etc. As used herein, a given percentage of identity with respect to a specified subject sequence, or a specified portion thereof, may be defined as the percentage of amino acids in the candidate derivative sequence identical with the amino acids in the subject sequence, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the Smith Waterman algorithm (Smith & Waterman, J. Mol. Biol. 147: 195-7 (1981)) using the BLOSUM substitution matrices (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9 (1992)) as similarity measures. A “% identity value” is determined by the number of matching identical amino acids divided by the sequence length for which the percent identity is being reported.
The term gp120 or gp41 ligand as used herein refers to a molecule (e.g., small molecule, peptide, antibody or antigen-binding fragment thereof, etc., either synthetic or natural) that binds to gp120 and/or gp41. In an embodiment, the gp120 or gp41 ligand is a synthetic molecule.
In an embodiment, the gp120 ligand is a small CD4 mimetic (CD4mc). The term “small CD4 mimetic” or “CD4mc” as used herein refers to molecules (e.g., small molecules, peptides, etc.) that bind in the Phe-43 cavity of gp120 and promote the transition of the Env protein to the “open”, CD4-bound conformation. Several CD4mc are known in the art and include, for example, NBD-556, NBD-557, DMJ-I-228, JP-III-48, M48U1 and BNM-III-170. CD4mc are also disclosed in PCT publication No. WO2013/090696 (see
Methods of determining if a HIV-1 Env trimer is in the prefusion closed state 1 conformation include (but are not limited to) negative stain cryogenic electron microscopy, smFRET (Munro et al., Science 2014, 346(6210):759-63) and antibody binding assays using a prefusion mature closed conformation specific antibody, such as VRC26, PGT128, PG9, PGT145, and derivatives thereof, which are well known in the art. Methods of determining if a HIV-1 Env ectodomain trimer is in the CD4-bound open state 2/3 conformation are also provided herein, and include (but are not limited to) negative stain cryogenic electron microscopy and antibody binding assays using a CD4-bound open conformation specific antibody, such as 17b or 19b (available, e.g., from the NIH AIDS Reagent Program, Cat. Nos. 4091 and 11436) which binds to a CD4-induced epitope.
In an embodiment, the agent that induces a state 1 configuration is a conformational blocker.
In an embodiment, the agent that induces a state 1 configuration is one of the HIV fusion inhibitors disclosed in Herschhorn et al., Nat Chem Biol. 2014; 10(10): 845-852, for example one of the following compounds:
In a further embodiment, the agent is compound 18A.
In an embodiment, the agent that induces a state 1 configuration is one of the HIV fusion inhibitors disclosed in Herschhorn et al., Nat Commun. 2017; 8: 1049, for example one of the following compounds:
In a further embodiment, the agent is compound 484.
In another embodiment, the agent is one of the compounds disclosed in U.S. Pat. Nos. 7,745,625, 8,168,615, 8,461,333 and 8,871,771 and in PCT publication No. WO 2005/090367. Representative examples of such compounds are depicted in
In a further embodiment, the agent is temsavir (BMS-626529)
or its prodrug fostemsavir (BMS-663068)
In an embodiment, the composition further comprises a carrier or excipient, in a further embodiment a pharmaceutically acceptable carrier or excipient. Such compositions may be prepared in a manner well known in the pharmaceutical art by mixing the antibody or an antigen-binding fragment thereof having a suitable degree of purity with one or more optional pharmaceutically acceptable carriers or excipients (see Remington: The Science and Practice of Pharmacy, by Loyd V Allen, Jr, 2012, 22nd edition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients, by Rowe et al., 2012, 7th edition, Pharmaceutical Press). The carrier/excipient can be suitable for administration of the antibody or an antigen-binding fragment thereof by any conventional administration route, for example, for oral, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration.
An “excipient” as used herein has its normal meaning in the art and is any ingredient that is not an active ingredient (drug) itself. Excipients include for example binders, lubricants, diluents, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents and other components. “Pharmaceutically acceptable excipient” as used herein refers to any excipient that does not interfere with effectiveness of the biological activity of the active ingredients and that is not toxic to the subject, i.e., is a type of excipient and/or is for use in an amount which is not toxic to the subject. Excipients are well known in the art, and the present system is not limited in these respects. In certain embodiments, one or more formulations of the dosage form include excipients, including for example and without limitation, one or more binders (binding agents), thickening agents, surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants/dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof. As those of skill would recognize, a single excipient can fulfill more than two functions at once, e.g., can act as both a binding agent and a thickening agent. As those of skill will also recognize, these terms are not necessarily mutually exclusive. Examples of commonly used excipient include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or auxiliary substances, such as emulsifying agents, preservatives, or buffers, which increase the shelf life or effectiveness.
In an embodiment, the composition further comprises a vaccine adjuvant. The term “vaccine adjuvant” refers to a substance which, when added to an immunogenic agent such as an antigen, non-specifically enhances or potentiates an immune response to the agent in the host upon exposure to the mixture. Suitable vaccine adjuvants are well known in the art and include, for example: (1) mineral salts (aluminum salts such as aluminum phosphate and aluminum hydroxide, calcium phosphate gels), squalene, (2) oil-based adjuvants such as oil emulsions and surfactant based formulations, e.g., incomplete or complete Freud's adjuvant, MF59 (microfluidised detergent stabilised oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21), (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), AS04 ([SBAS4] aluminum salt with MPL), ISCOMS (structured complex of saponins and lipids), polylactide co-glycolide (PLG), (4) microbial derivatives (natural and synthetic), e.g., monophosphoryl lipid A (MPL), Detox (MPL+M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoidal immunostimulators able to self-organize into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified LT and CT (genetically modified bacterial toxins to provide non-toxic adjuvant effects), complete Freud's adjuvant (comprising inactivated and dried mycobacteria) (5) endogenous human immunomodulators, e.g., hGM-CSF or hIL-12 (cytokines that can be administered either as protein or plasmid encoded), Immudaptin (C3d tandem array) and/or (6) inert vehicles, such as gold particles.
In an embodiment, the mutated HIV-1 gp120 polypeptide or composition may be comprised in a cell, a liposome or a virus-like particle (VLP). Thus, in another aspect, the present disclosure provides a cell, liposomes (see, e.g., Rao et al., J Infect Dis. 2018; 218(10):1541-1550) or VLP expressing at its surface the mutated HIV-1 gp120 polypeptide disclosed herein, for example in the form of a trimer with gp41. VLPs are multimeric nanostructures morphologically resembling authentic viral particles composed of viral structural proteins with inherent self-assembly properties but are devoid of viral genetic materials. The display of HIV Env trimers at the surface of VLPs is considered a promising strategy for eliciting an immune response (e.g., neutralizing antibodies) against HIV (Zhao et al., Vaccines (Basel). 2016; 4(1): 2).
In an embodiment, the mutated HIV-1 gp120 polypeptide may be delivered in the form of a nucleic acid comprising a sequence encoding the mutated HIV-1 gp120 polypeptide. The nucleic acid may be optimized, such as by codon optimization, for expression in a targeted mammalian subject (e.g., human). As discussed below, the nucleic acid may be incorporated into a vector (e.g., a viral vector, such as an adenovirus or poxvirus vector). Accordingly, the composition or vaccine disclosed herein may include one or more of these vectors. The mutated HIV-1 gp120 polypeptide may be recombinantly expressed in a cell or organism, or may be directly administered to a subject (e.g., a human) infected with, or at risk of becoming infected with, HIV (e.g., HIV-1).
The present disclosure also provides vectors including the nucleic acid molecule encoding the mutated HIV-1 gp120 polypeptide. The vector can be, for example, a carrier (e.g., a liposome), a plasmid, a cosmid, a yeast artificial chromosome, or a virus (e.g., an adenovirus vector or a poxvirus vector) that comprises the nucleic acid molecule encoding the mutated HIV-1 gp120 polypeptide.
The adenovirus vector may be derived from a recombinant adenovirus serotype 11 (Ad11), adenovirus serotype 15 (Ad15), adenovirus serotype 24 (Ad24), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), adenovirus serotype 49 (Ad49), adenovirus serotype 50 (Ad50), Pan9 (AdC68), or a chimeric variant thereof (e.g., adenovirus serotype 5 HVR48 (Ad5HVR48)). The poxvirus vector may be derived, for example, from modified vaccinia virus Ankara (MVA). These vectors can include additional nucleic acid sequences from several sources.
Such vectors may be constructed using any recombinant molecular biology technique known in the art. The vector, upon transfection or transduction of a target cell or organism, can be extrachromosomal or integrated into the host cell chromosome. The nucleic acid component of a vector can be in single or multiple copy number per target cell, and can be linear, circular, or concatamerized. The vectors can also include internal ribosome entry site (IRES) sequences to allow for the expression of multiple peptide or polypeptide chains from a single nucleic acid transcript (e.g., a polycistronic vector, e.g., a bi- or tri-cistronic vector).
Vectors may also include gene expression elements that facilitate the expression of the encoded mutated HIV-1 gp120 polypeptide. Gene expression elements include, but are not limited to, (a) regulatory sequences, such as viral transcription promoters and their enhancer elements, such as the SV40 early promoter, Rous sarcoma virus LTR, and Moloney murine leukemia virus LTR; (b) splice regions and polyadenylation sites such as those derived from the SV40 late region; and (c) polyadenylation sites such as in SV40. Also included are plasmid origins of replication, antibiotic resistance or selection genes, multiple cloning sites (e.g., restriction enzyme cleavage loci), and other viral gene sequences (e.g., sequences encoding viral structural, functional, or regulatory elements, such as the HIV long terminal repeat (LTR)).
To improve the delivery of the nucleic acid into a cell or subject in order to promote formation of the Env trimers, lipoplexes (e.g., liposomes) and polyplexes can be used to protect the nucleic acid from undesirable degradation during the transfection process. The nucleic acid molecules can be covered with lipids (e.g., cationic lipids) in an organized structure like a micelle or a liposome. When the organized structure is complexed with the nucleic acid molecule it is called a lipoplex. Cationic lipids, due to their positive charge, naturally complex with the negatively-charged nucleic acid, and are thus preferred for such liposomes. Polyplexes refer to complexes of polymers with nucleic acids.
Exemplary cationic lipids and polymers that can be used in combination with one or more of the nucleic acid molecules encoding mutated HIV-1 gp120 polypeptide to form lipoplexes or polyplexes include, but are not limited to, polyethylenimine, lipofectin, lipofectamine, polylysine, chitosan, trimethylchitosan, and alginate.
In another aspect, the present disclosure provides a method for eliciting an immune response to HIV-1 in a subject, comprising administering to the subject a prophylactically or therapeutically effective amount of (i) the mutated HIV-1 gp120 polypeptide defined herein, and (ii) a gp120 or gp41 ligand. In an embodiment, the composition defined herein is administered.
In another aspect, the present disclosure provides the use of (i) the mutated HIV-1 gp120 polypeptide defined herein, and (ii) a gp120 or gp41 ligand, for eliciting an immune response to HIV-1 in a subject. In an embodiment, the composition defined herein is used.
In another aspect, the present disclosure provides the use of (i) the mutated HIV-1 gp120 polypeptide defined herein, and (ii) a gp120 or gp41 ligand, for the manufacture of a medicament for eliciting an immune response to HIV-1 in a subject. In an embodiment, the composition defined herein is used.
In another aspect, the present disclosure provides a combination comprising (i) the mutated HIV-1 gp120 polypeptide defined herein, and (ii) a gp120 or gp41 ligand for eliciting an immune response to HIV-1 in a subject. In an embodiment, the combination is present in the composition defined herein.
When treating disease (e.g., HIV infection/AIDS), the mutated HIV-1 gp120 polypeptide and gp120 or gp41 ligand, combination or composition disclosed herein may be administered to the subject either before the occurrence of symptoms or a definitive diagnosis or after diagnosis or symptoms become evident. For example, the composition may be administered, for example, immediately after diagnosis or the clinical recognition of symptoms or 2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, or even 3, 4, or 6 months after diagnosis or detection of symptoms. In an embodiment, the mutated HIV-1 gp120 polypeptide and gp120 or gp41 ligand, combination or composition disclosed herein is administered to a subject that is not infected by HIV, e.g., as a prophylactic vaccine to confer immune protection (partial or complete) against future HIV-1 infections, for example a subject at-risk of being infected. In an embodiment, the mutated HIV-1 gp120 polypeptide and gp120 or gp41 ligand, combination or composition disclosed herein is administered to a subject that is already infected by HIV, e.g., as a therapeutic vaccine to boost the immune response against HIV-1 and reduce viral load.
The mutated HIV-1 gp120 polypeptide and gp120 or gp41 ligand, combination or composition disclosed herein may be administered in combination with one or more additional therapeutic agents, for example, for preventing or treating an HIV infection (e.g., an HIV-1 infection) in a subject. Such additional therapeutic agents can include, for example, a broadly neutralizing antibody (bnAb), e.g., those described in PCT publications No. WO2015/048770, WO 2012/030904, and WO 2013/055908. Exemplary bnAbs that can be administered in combination with the compositions of the invention include PGT121, PGT122, PGT123, PGT124, PGT125, PGT126, PGT127, PGT128, PGT130, PGT131, PGT132, PGT133, PGT134, PGT135, PGT136, PGT137, PGT138, PGT139, PGT141, PGT142, PGT143, PGT144, PGT145, PGT151, PGT152, PGT153, PGT154, PGT155, PGT156, PGT157, PGT158, 3BNC117 and 10-1074, a derivative or clonal relative thereof, or a combination thereof.
The additional therapeutic agent may also be an antiretroviral therapy (ART), which may, e.g., be selected from any one or more of the following, or combinations thereof: efavirenz, emtricitabine, and tenofovir disoproxil fumarate (Atripla); emtricitabine, rilpivirine, and tenofovir disoproxil fumarate (Complera); elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate (Stribild); lamivudine and zidovudine (Combivir); emtricitabine, FTC (Emtriva); lamivudine, 3TC (Epivir); abacavir and lamivudine (Ebzicom); zalcitabine, dideoxycytidine, ddC (Hivid); zidovudine, azidothymidine, AZT, ZDV (Retrovir); abacavir, zidovudine, and lamivudine (Trizivir); tenofovir disoproxil fumarate and emtricitabine (Truvada); enteric coated didanosine, ddl EC (Videx EC); didanosine, dideoxyinosine, ddl (Videx); tenofovir disoproxil fumarate, TDF (Viread); stavudine, d4T (Zerit); abacavir sulfate, ABC (Ziagen); Rilpivirine (Edurant); Etravirine (Intelence); delavirdine, DLV (Rescriptor); efavirenz, EFV (Sustiva); nevirapine, NVP (Viramune or Viramune XR); amprenavir, APV (Agenerase); tipranavir, TPV (Aptivus); indinavir, IDV (Crixivan); saquinavir (Fortovase); saquinavir mesylate, SQV (Invirase); lopinavir and ritonavir, LPV/RTV (Kaletra); Fosamprenavir Calcium, FOS-APV (Lexiva); ritonavir, RTV (Norvir); Darunavir (Prezista); atazanavir sulfate, ATV (Reyataz); nelfinavir mesylate, NFV (Viracept); enfuvirtide, T-20 (Fuzeon); maraviroc (Selzentry); raltegravir, RAL (Isentress); and dolutegravir (Tivicay).
The additional therapeutic agent can also be an immunomodulator. The immunomodulator may be selected, e.g., from any one or more of the following, or combinations thereof: AS-101, Bropirimine, Acemannan, CL246,738, EL10, FP-21399, Gamma Interferon, Granulocyte Macrophage Colony Stimulating Factor, HIV Core Particle Immunostimulant, IL-2, Immune Globulin Intravenous, IMREG-1, IMREG-2, Imuthiol Diethyl Dithio Carbamate, Alpha-2 Interferon, Methionine-Enkephalin, MTP-PE, Muramyl-Tripeptide, Granulocyte Colony Stimulating Factor, Remune, CD4 (e.g., recombinant soluble CD4), rCD4-IgG hybrids, SK&F106528 Soluble T4, Thymopentin, Tumor Necrosis Factor, and Infliximab.
The additional therapeutic agent can also be a reservoir activator. The reservoir activator may be selected, e.g., from any one or more of the following, or combinations thereof: histone deacytelase (HDAC) inhibitors (e.g., romidepsin, vorinostat, and panobinostat), immunologic activators (e.g., cytokines and TLR agonists), and dedicated small molecule drugs.
Administration of an additional therapeutic agent may be prior to, concurrent with, or subsequent to the administration of the composition or vaccine disclosed herein.
In another aspect, the present disclosure relates to a method for determining whether a test agent (e.g., an antibody) binds to an HIV Env trimer into an open (state 2/3) conformation comprising contacting said test agent with the mutated HIV-1 gp120 polypeptide defined herein, and a gp120 ligand capable of inducing an open (state 2/3) conformation, e.g., a CD4mc.
In another aspect, the present disclosure relates to a method (e.g., in vitro) for determining whether a test agent (e.g., an antibody) binds to an HIV Env trimer into a closed (state 1) conformation comprising contacting said test agent with the mutated HIV-1 gp120 polypeptide defined herein, and a gp120 ligand capable of inducing a closed (state 1) conformation, e.g., a conformational blocker.
In another aspect, the present disclosure relates to a method (e.g., in vitro) for inducing an HIV Env trimer into an open (state 2/3) conformation comprising contacting an HIV Env trimer comprising the mutated HIV-1 gp120 polypeptide defined herein with a gp120 ligand capable of inducing an open (state 2/3) conformation, e.g., a CD4mc.
In another aspect, the present disclosure relates to the use of the mutated HIV-1 gp120 polypeptide defined herein with a gp120 ligand capable of inducing an open (state 2/3) conformation, e.g., a CD4mc, for inducing an HIV Env trimer into an open (state 2/3) conformation, or for the manufacture of a medicament for inducing an HIV Env trimer into an open (state 2/3) conformation.
In another aspect, the present disclosure relates to a method (e.g., in vitro) for inducing an HIV Env trimer into a closed (state 1) conformation comprising contacting an HIV Env trimer comprising the mutated HIV-1 gp120 polypeptide defined herein with a gp120 ligand capable of inducing a closed (state 1) conformation, e.g., a conformational blocker.
In another aspect, the present disclosure relates to the use of the mutated HIV-1 gp120 polypeptide defined herein with a gp120 ligand capable of inducing a closed (state 1) conformation, e.g., a conformational blocker, for inducing an HIV Env trimer into a closed (state 1) conformation, or for the manufacture of a medicament for inducing an HIV Env trimer into a closed (state 1) conformation.
In another aspect, the present disclosure relates to a method for determining whether a test agent induces a closed (state 1) conformation of an HIV Env trimer comprising (a) contacting the mutated HIV-1 gp120 polypeptide defined herein with said test agent, and (b) determining whether the HIV Env trimer is in a closed (state 1) conformation.
In another aspect, the present disclosure relates to a method for determining whether a test agent induces an open (state 2/3) conformation of an HIV Env trimer comprising (a) contacting the mutated HIV-1 gp120 polypeptide defined herein with said test agent, and (b) determining whether the HIV Env trimer is in an open (state 2/3) conformation.
Such determining may be performed using assays capable of measuring conformational changes of membrane-bound trimeric Env, for example, antibodies that specifically binds to the closed (state 1) or open (state 2/3) conformation, as described above, or the assay described in Veillette M et al., 2014. J Vis Exp doi:10.3791/51995:51995 or Haim H et al., PLoS Pathog 7:e1002101.
The present invention is illustrated in further details by the following non-limiting examples.
All HIV-1 sequences have been analyzed together or segregated by clades using the NIH Los Alamos HIV database to determine the degree of conservation of residues located in layer 1 (residue 61), layer 2 (residues 105 and 108), layer 3 (residues 474, 475 and 476) (collectively named LM for layer mutants) and the Phe43 cavity residue 375. By comparing consensus sequences from CRF01_AE strains with the different HIV-1 group M clades (clades A to K), it is possible to see the divergence between CRF01_AE and all other clades (
CD4mc were used as probes to evaluate the potential impact of the LM residues on shaping the Phe43 cavity. First, the effect of the H375S mutation on the sensitivity of two CRF01_AE isolates (tier 1 92TH023 and tier 2 CM244) to neutralization by different CD4 mimics including soluble CD4 (sCD4), CD4mc (BNM-III-170) and a CD4 miniprotein (M48U1) was assessed. Replacement of histidine by a serine at position 375 (H375S) into both HIV-1CRF01_AE Envs completely abolished the susceptibility of pseudotypes tosCD4 neutralization (
To gain a better understanding of the impact that the LM and 375 changes have on Env conformation, an assay to measure sCD4 and BNM-III-170 capacity to interact with Env was developed. Briefly, CD4-negative cells were transfected with Env variants. Two days post-transfection, BNM-III-170 or the vehicle (DMSO) was added to Env-expressing cells. The impact on Env conformation was detected by evaluating binding of broadly-neutralizing antibodies (bNAbs) that preferentially recognize the “closed” state 1 trimer (3BNC117, NIH45-46 G54W, PG16, PGT121 and PGT128), non-neutralizing (nnAbs) CD4i Abs (17b, 19b, F240 and A32) or soluble CD4 (sCD4) that preferentially recognize the “open” state 2/3 Env conformation (Munro J B, et al., 2014. Science 346:759-763; Lu M, et al. 2019. Nature 568:415-419; Derking R. et al. 2015. PLoS Pathog 11: e1004767; Ma X. et al. 2018, Elife 7; Alsahafi N. et al. 2019. Cell Host Microbe 25:578-587 e575). Their ability to interact with Env was obtained by calculating the decrease of these ligands binding compared to DMSO. Overall, the results presented in
To assess whether the LM+HS or LM+HT mutations were restricted to inhibitors “pushing” Env to more “open” (state 2/3) conformations or if they could enhance Env flexibility in such a way that they could respond to inhibitors “pushing” in opposite directions (to a closed, state 1 conformations), their susceptibility to conformational blockers (BMS-626529 and 484) known to stabilize Env state 1 (10, 16, 17) was tested. Strikingly, these unique set of mutations sensitized CRF01_AE Env to neutralization to BMS-626529 and 484 (
Overall, the studies described herein reveal a complex interplay between the gp120 inner domain and the Phe43 cavity that could be exploited to guide the development of more potent Env inhibitors (CD4mc and conformational blockers), help expose the CD4 binding site and the elusive state 1 conformation, all properties highly thought to develop an efficient HIV-1 vaccine.
Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise.
The present application claims the benefit of U.S. provisional patent application No. 62/904,821 filed Sep. 24, 2019, which is incorporated herein by reference.
This invention was made with Government support under NIH Grant Nos. R01A1129769 and Contract No. P016M56550/A1150741. The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2020/051274 | 9/23/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62904821 | Sep 2019 | US |
