Polypeptide constructs with potent Anti-HIV activity

Abstract

Polypeptide constructs directed against the CD4+ T-cell surface proteins and HIV Env proteins are disclosed. These constructs may be used to treat and prevent HIV infection and to prevent the entry of HIV into mammalian cells.

Description

POLYPEPTIDE CONSTRUCTS WITH POTENT ANTI-HIV ACTIVITYCROSS-REFERENCE TO RELATED APPLCATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 63 / 563,741, filed March 11, 2024. The entire disclosure of U.S. Provisional Application Serial No. 63 / 563,741 is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure provides polypeptide constructs that bind to and interfere with CD4+ T-cell surface proteins and HIV viral proteins that together participate in HIV entry into human T cells, and methods of using these polypeptide constructs to treat and prevent HIV infection.SEQUENCE LISTING SUBMITTED AS A .XML FILE VIA EFS-WEB

[0003] This application contains a Sequence Listing submitted as an XML file named “10439NIDDK-l-PCT_Sequence_Listing.xml” having a size in bytes of 23,012 bytes and created on March 10, 2025. The information contained in this electronic file is hereby incorporated by reference herein in its entirety pursuant to 37 CFR § 1.52(e)(5).BACKGROUND

[0004] If left untreated, infection with the Human Immunodeficiency Virus (HIV) leads to death of the infected person. HIV infects CD4+ T-cells, leading to a precipitous decline in the number of CD4+ T-cells in the infected person. When CD4+ T-cell numbers decline below a critical level, cell-mediated immunity is effectively lost and infections with a variety of opportunistic microbes appear, resulting in Acquired Immunodeficiency Syndrome (AIDS). Unable to defend against these opportunistic infections, the patient will ultimately succumb to one of these infections.

[0005] To enter a host cell, HIV must attach to the surface of the host cell and interact with two separate receptors on the cell surface: the CD4 receptor and a coreceptor, either CXCR4 or CCR5. The HIV envelope glycoprotein (Env) is a trimer of heterodimers consisting of gpl20 and gp41, with gpl20 being more variable than gp41. CD4 is the primary receptor of the HIV Env glycoprotein 120 (gpl20) and is critical for HIV entry into host cells.

[0006] HIV infection is initiated by the binding of gpl20 to CD4, which induces a series of conformational changes in gpl20 that consequently exposes its third variable (V3) loop for specific recognition of a coreceptor, either CCR5 (in the R5- or M-tropic virus) or CXCR4 (in the X4- or T-tropic virus). The interaction of viral gp!20 with a coreceptor causes the gp 120-gp41 complex to undergo a conformational change that leads to the formation of a trimeric hairpin structure of gp41, enabling the viral envelope to fuse with the host cell membrane, resulting in the release of the viral capsid into the cytoplasm of the host T-cell.

[0007] Currently there is no cure available for HIV / AIDS, but HIV infected persons can suppress replication of the virus through a variety of anti-retroviral therapies (ART). ART consists of the administration of a cocktail of multiple anti-viral compounds. However, because HIV readily mutates, the virus often becomes resistant to one or more compounds in the ART cocktail. Additionally, ART is associated with a number of side effects. While antiretroviral adherence is the second strongest predictor of progression to AIDS and death, after CD4 count, incomplete adherence to ART is common in all groups of treated individuals. The average rate of adherence to ART is approximately 70%, despite the fact that long-term viral suppression requires near-perfect adherence. The resulting treatment failure diminishes long-term clinical success and increases the risk of drug resistance.

[0008] Neutralizing antibodies or immunoglobulin fragments have also been designed and tested as a means of treating and preventing HIV / AIDS (see, US 2017 / 0247435A1; US 2018 / 0179299A1; US 2019 / 0292267 Al; US 2022 / 0002391A1). Some of these immunological molecules were directed against intra-cellular HIV proteins, necessitating intracellular expression of the immune protein, and in most cases, resistance against the immunological molecule / therapeutic eventually developed (Fessel et al., 2011 Antiviral Res 92:484-87). Thus, effective therapies for the treatment of HIV infection are still needed, in particular, therapies that can overcome the resistance that quickly develops against ART.SUMMARY

[0009] This invention relates, in part, to polypeptide constructs that bind with and prevent or interfere with the interaction between the HIV envelope spike protein and T-cell surface proteins that interact together to guide HIV entry into CD4+ T-cells.

[0010] A polypeptide construct of this disclosure may comprise a nanobody (Nb) linked to a Human Immunodeficiency Virus (HIV) fusion-inhibitor (FI) peptide. In these polypeptide constructs, the Nb may bind to a T-cell surface protein. The T-cell surface protein may be a CD4 receptor, or a CXCR4 coreceptor, or a major histocompatibility complex (MHC) protein, such as an MHC class 1 (MHC-1) protein.

[0011] Exemplary nanobodies that are useful in the polypeptide constructs of this disclosure include, but are not limited to, a Nb selected from the group consisting of: Nb-3F11 (SEQ ID NO:4), Nb-CD4-bl (SEQ ID NO:8), Nb-281E10 (SEQ ID NO:5), Nb-281F12 (SEQ ID NO:6), and Nb-30 (SEQ ID NO:7).

[0012] In these polypeptide constructs, the Nb may bind to an HIV protein, such as an envelope (Env) protein. These nanobodies may bind to an Env protein domain selected from CD4bs (CD4 binding site) domain, V3 loop domain, gp41 heptad domain, and MPER (membrane proximate external region) domain. These nanobodies may bind to one or both of the Env proteins, gp41 and gp 120.

[0013] In these polypeptide constructs, the HIV Fusion Inhibitor (FI) peptide may bind with an HIV protein. In these polypeptide constructs, the HIV protein may be an envelope (Env) protein such as gp41 or gpl20. The FI peptide may bind with a gp41 protein domain selected from the N-terminal heptad repeat of gp41 , the fusion peptide domain of gp41, and the C-terminal heptad repeat of gp41.

[0014] Exemplary peptides useful in the polypeptide constructs of this disclosure include, but are not limited to, a peptide selected from C34 (SEQ ID NO:9) and N36 (SEQ ID NO: 12).

[0015] In these polypeptide constructs, the Nb and the peptide may be linked through a covalent linker. The covalent linker may comprise amino acid residues, which may, for example, comprise at least two or at least three glycine amino acid residues, or variable sequences of one or more neutral amino acids, such as glycine, serine, alanine, lysine, glutamate. In these constructs, the linker may comprise an amino acid sequence such as LPETGGG (SEQ ID NO: 13); LPETGGGHHHHHH (SEQ ID NO: 14); GGSGGGS (SEQ ID NO: 15); GGSGGGSGGSGGGS (SEQ ID NO: 16); EAAAKEAAAKEAAAK (SEQ ID NO: 17); or GGGGSGGGGSGGGGS (SEQ ID NO: 18). Alternatively or additionally, the linker may comprise one or more small molecules. For example, the linker may comprise one or more of an azide, a triazole, an azide-derivatized dibenzo-azacyclooctyne (DBCO), andpolyethylene glycol (PEG). An exemplary linker useful in the polypeptide constructs of this disclosure is an azide-derivatized dibenzo-azacyclooctyne (DBCO) linked to an LPETGG peptide.

[0016] This disclosure also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and any one or more of the polypeptide constructs of this disclosure.

[0017] This disclosure also provides methods of treating a subject infected with HIV by administering to the subject at least one of these polypeptide constructs, or a pharmaceutical composition comprising one or more of these polypeptide constructs. In these methods of treatment, the administration may be effective to treat Acquired Immunodeficiency Syndrome (AIDS) in the subject. In these methods, the administration may effective to inhibit or completely block viral entry of HIV into T-cells of the subject, and / or to increase white blood cell count in the treated subject, and / or to increase in the number of CD4+ T- cells in the subject, and / or to decrease the HIV-titer in the subject, and / or to decrease opportunistic microbial infections in the subject. Preferably, in these methods the administration causes only minimal undesirable side effects in the subject that are typically associated with other anti-HIV treatment regimens.

[0018] This disclosure also provides methods of preventing an HIV infection in an uninfected subject by administering to the uninfected subject at least one of these polypeptide constructs, or a pharmaceutical composition comprising one or more of these polypeptide constructs. In these methods of prevention, the polypeptide construct may prevent HIV infection in a subject who is HIV-negative, and / or may inhibit HIV fusion with CD4+ and / or CXCR4+ cells of the subject.

[0019] This disclosure also provides methods of inhibiting the onset or progression of HIV infections or HIV-associated disorder in a subject by administering to the uninfected subject at least one of these polypeptide constructs, or a pharmaceutical composition comprising one or more of these polypeptide constructs. In these methods, the administered polypeptide construct may inhibit fusion of HIV to CXCR4+ and / or CD4+ immune cells in the subject.

[0020] This Summary is neither intended nor should it be construed as representative of the full extent and scope of the present disclosure. Moreover, references made herein to “this disclosure,” “the present disclosure,” or aspects thereof, should be understood to mean certain embodiments of the present invention and should not necessarily be construed aslimiting all embodiments to a particular description. The present invention is set forth in various levels of detail in this Summary as well as in the attached drawings and the Detailed Description and no limitation as to the scope of the present invention is intended by either the inclusion or non-inclusion of elements, components, etc. in this Summary. Additional aspects of the present invention will become more readily apparent from the Detailed Description, particularly when taken together with the figures.BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1 shows an exemplary stained SDS-PAGE gel run with seven nanobodies of this disclosure demonstrating purity of the synthesized proteins.

[0022] FIG. 2A shows the FACS counts for synthesized nanobodies of this disclosure reflecting binding affinity for three TZM-bl cell surface proteins. FIG. 2B shows the FACS counts for synthesized nanobodies of this disclosure reflecting binding affinity for two HEK293 T-cell surface proteins.

[0023] FIG. 3A shows the FACS counts for the CD4-targeting nanobody Nb-3F11 (SEQ ID NO:4) and FIG. 3B shows the FACS counts for the CD4-targeting nanobody Nb-CD4-bl (SEQ ID NO: 8), indicating detectable binding of the CD4 receptor protein at concentrations down to 0.1 nM.

[0024] FIG. 4 is a graph quantifying the binding data from the binding competition assays conducted between two CD4 receptor-binding nanobodies, Nb-3F11 (SEQ ID NO:4) and Nb-CD4bl(SEQ ID NO: 8).

[0025] FIG. 5 is a schematic representation of one means of synthesizing polypeptide constructs of this disclosure. The synthesis scheme shows a sortagged nanobody, including a His-tag (LPETGGHHHHHH; SEQ ID NO: 14), used to form a linker (LPETGGG; SEQ ID NO: 13) between the C-terminus of the nanobody and the N-terminus of the peptide.

[0026] FIG. 6 is a schematic representation of another means of synthesizing polypeptide constructs of this disclosure, showing a sortagged nanobody, including a His-tag (LPETGGHHHHHH; SEQ ID NO: 14), and the formation of a linker (LPETGGG-triazole; SEQ ID NO: 13) between the C-terminus of the nanobody and the C-terminus of the peptide.

[0027] FIG. 7 shows an exemplary stained SDS-PAGE gel run with six nanobody-FI peptide constructs of this disclosure demonstrating purity of the synthesized polypeptide constructs.

[0028] FIG. 8 A shows the HIV neutralization curve against the YU2 strain of HIV- 1 pseudoviruses for the C34 fusion inhibitor peptide (G3-C34-Cys; SEQ ID NOTO) and four nanobody constructs of this disclosure. FIG. 8B shows another neutralization curve for the same constructs depicted in FIG. 8 A, with the exception that nanobody 281E10 was replaced by 28 IF 12. FIG. 8C shows another neutralization curve that used two individual nanobodies [Nb-3F11 (SEQ ID NO:4) and Nb-281F12 (SEQ ID NO: 6)] and polypeptide constructs of this disclosure. FIG. 8D shows another neutralization curve that tested one nanobody - peptide construct (Nb-30-C34), one antiviral peptide (EP417), and four individual nanobodies.

[0029] FIG. 9 shows the HIV neutralization curve against the YU2 strain of HIV- 1 pseudoviruses for five nanobody constructs of this disclosureDETAILED DESCRIPTION

[0030] As initially described in Hamers-Casterman et al., 1992, Camelids, which include the Old World species camels and dromedaries, and the New World species llamas, alpacas, and vicunas, produce heavy chain-only antibodies. The single-domain variable fragments of these heavy chain-only antibodies are referred to as single-domain antibodies (sdAbs) or variable domain of the heavy chain of HCAbs (“VHH”) or “nanobodies” (Nbs). The designation “nanobody” for camelid single chain variable regions was originally claimed as a brand name for what is more accurately called VHH, but the term nanobody has come into general usage interchangeably with VHH. Nanobodies retain the immunoglobulin fold shared by antibodies, using three hypervariable loops, CDR1, CDR2, and CDR3, to bind to their targets. Many nanobodies bind to their targets with affinities similar to conventional full-size antibodies, but possess other properties superior to antibodies. For example, because nanobodies are small proteins of approximately 12-15 kDa, yet possess the binding specificity and affinity of antibodies, they have many uses in biotechnology and medicine. Typically, a nanobody has an amino acid sequence with the general structure: FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively.

[0031] The present invention relates to polypeptide constructs, also referred to as “polypeptide constructs of this disclosure” that comprise or, in some embodiments, consist essentially of:(1) a first building block that is a nanobody (Nb, sdAbs, ISVD, or VHH) that specifically binds a T-cell surface protein or an HIV Env protein, for example a CD4 receptor or a CXCR4 coreceptor on the surface of a T-cell, or a gp41 and / or gpl20 components of the HIV Env protein; and (2) a second building block that is a peptide (fusion inhibitor peptide or FI peptide) that binds with an HIV surface protein, for example, the HIV envelope glycoprotein (Env), including, for example, the gpl20 and / or gp41 components of HIV Env. The first and second building blocks are covalently linked through a linker, for example an amino acid sequence or a small organic molecule, or combinations of these linking molecules.

[0032] The inventors demonstrated that binding by the polypeptide constructs of this disclosure, comprising the first (nanobody) and second (peptide) building blocks described above, to a CD4+ T-cell surface protein and the HIV Env protein, respectively, produces a synergy between the two building blocks in neutralizing HIV (see Example 2, infra). Additionally, the inventors have surprisingly discovered these polypeptide constructs are more effective than combinations of the two individual moieties (nanobodies and peptides) (see Example 2, infra). “Synergy” between two or more agents refers to the combined effect of the agents which is greater than their additive effects. Agents may be peptides, proteins, such as nanobodies, and drug forms thereof.

[0033] The polypeptides of the invention are designed to inhibit HIV infection. “HIV” refers to the human immunodeficiency virus. HIV includes HIV-1 and HIV-2. HIV-1 includes, but is not limited to, extracellular virus particles and the forms of HIV-1 associated with HIV-1 infected cells. The human immunodeficiency virus (HIV) may be either of the two known types of HIV (HIV-1 or HIV-2). The HIV-1 virus may represent any of the known major subtypes (classes A, B, C, D, E, F, G, H, or J), outlying subtype (Group 0), or an as yet to be determined subtype of HIV- 1.

[0034] “HIV infection” refers to the entry of HIV into a susceptible cell. As described above, infection of cells by human immunodeficiency virus type 1 (HIV-1) is mediated by the viral envelope (Env) glycoproteins gpl20 and gp41, which are expressed as a non-covalent, oligomeric complex on the surface of the virus and virally infected cells. Entry of the virus into target cells proceeds through a cascade of events at the cell surface that include (1) high-affinity interaction between the HIV surface glycoprotein gpl20 to the cell surfacereceptor CD4, (2) Env binding to fusion coreceptors, and (3) conformational changes in the viral transmembrane glycoprotein gp41, which mediates fusion of the viral and cellular membranes. Inhibiting HIV infection to T-cells relates to inhibiting at least one function in this HIV T-cell fusion and entry cycle. These functions include, for instance, binding of HIV to the receptor CD4, binding of HIV to the coreceptor, and entry of HIV into a target cell. HIV-T-cell fusion is prevented or significantly reduced, which may ultimately prevent or inhibit the transmission of HIV. Inhibition of HIV infection can be measured by various methods, both in vitro and in vivo. Preferably, inhibiting HIV infection results in reducing the viral load or the maintenance of a reduced viral load and preferably by ameliorating the medical condition of the HIV infected subject. The term “viral load” refers to the amount of HIV particles in a sample of blood, generally indicated as the number of copies per milliliter of blood. For instance, a viral load of more than 100,000 copies / ml would be considered high, while a viral load of less than 10,000 copies / ml would be considered low. Preferably, the viral load is reduced to undetectable levels (i.e., <50 copies per ml).

[0035] Inhibition, as used herein, includes both complete and partial inhibition. Thus, this disclosure includes polypeptide constructs comprising a nanobody and a peptide that inhibit binding of HIV to CD4 and / or a co-receptor, such as CXCR4, by more than 1%, more than 2%, more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or up to 100% inhibition.

[0036] This disclosure includes polypeptide constructs that inhibit HIV infection by about 10%, 20%, 30%, 40%, 50%, 60%, 80%, 90% and preferably 95% or more, such as 100% (as measured in an HIV infection assay). Inhibition may also be expressed as the IC50 (Inhibitory Concentration), defined as the inhibitory concentration at which 50% of HIV is inhibited from binding to and infecting a T-cell. For the polypeptide constructs of this disclosure, the IC50 for inhibition of HIV binding to T-cells is lower than 500 pM, lower than 100 pM, lower than 50 pM, lower than 10 pM, lower than 50 pM, lower than 1 pM, lower than 500 nM, lower than 100 nM, lower than 90 nM, lower than 80 nM, lower than 70 nM, lower than 60 nM, lower than 50 nM, lower than 40 nM, lower than 30 nM, lower than 20 nM, lower than 10 nM, lower than 9 nM, lower than 8 nM, lower than 7 nM, lower than6 nM, lower than 5 nM, lower than 4 nM, lower than 3 nM, lower than 2 nM, or even lower than 1 nM.

[0037] The term “polypeptide” is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the amino acid sequence. Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms can be used interchangeably herein unless specifically indicated otherwise. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide can be an entire protein, or a subsequence thereof.

[0038] Exemplary nanobody and peptide sequences, and variants thereof, which may form the first and second building blocks of the polypeptide constructs of this disclosure, are set forth in SEQ ID NOS: 1-23, summarized in Table 1.

[0039] Table 1Nanobodies

[0040] Within the polypeptide constructs of this disclosure, the first of the two “building blocks” is a nanobody. The nanobody may specifically bind to a T-cell surface protein. Preferably, the T-cell surface protein is the CD4 receptor or the CXCR4 coreceptor. Alternatively, the nanobody may specifically bind to an HIV protein. Preferably, the HIV protein is the Env protein.

[0041] These nanobodies may be derived in any suitable manner and from any suitable source and may, for example, be naturally occurring amino acid sequencesfrom a suitable species of Camelid) or synthetic or semi-synthetic variable light or variable heavy chain immunoglobulin proteins (e.g., from humans). Such nanobodies may include “humanized” or otherwise “sequence optimized” VHHs. Humanized nanobodies may be made by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring nanobody sequence (in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a variable heavy domain from a typical four-chain human antibody.

[0042] Exemplary nanobodies that may be used as the first of the two “building blocks” in the peptide constructs of this disclosure include, but are not limited to: Nb-3F11 (SEQ ID NO:4) that specifically binds to the T-cell CD4 receptor, Nb-281E10 (SEQ ID NO: 5) that specifically binds to the T-cell CXCR4 co-receptor, Nb-281F12 (SEQ ID NO:6) that specifically binds to the T-cell CXCR4 co-receptor, Nb-CD4-bl (SEQ ID NO: 8) that specifically binds to the T-cell CD4 receptor, Nb-J3 (SEQ ID NO: 1) that specifically binds to HIV gp!20 Env protein, Nb-IFIO (SEQ ID NO:2) that specifically binds to V3 loop of HIV Env protein, and Nb-2E7 (SEQ ID NO:3) that specifically binds to heptad repeat of HIV gp41 Env protein.Peptides

[0043] Within the polypeptide constructs of this disclosure, the second of the two “building blocks” is a peptide. This peptide may be a fusion inhibitor (FI) peptide that binds with HIV Env proteins to disrupt the formation of the protein structure formed by the HIV and T-cell protein components resulting in fusion of the HIV viral envelope and the T-cell membrane, thereby preventing HIV-T-cell fusion and ultimately preventing HIV infection of the T-cell.

[0044] Exemplary peptides that may be used as the second of the two “building blocks” in the peptide constructs of this disclosure include, but are not limited to:C34 (SEQ ID NO: 11) that binds with the N-terminal heptad repeat of gp41, and N36 (SEQ ID NO: 12) that binds with the C-terminal heptad repeat of gp41.

[0045] Both the nanobody and the peptide “building blocks” of this disclosure may be synthesized utilizing techniques available to those skilled in the art. For example, DNA molecules encoding a nanobody or peptide may be constructed based on the coding sequence of the peptides using molecular cloning techniques. These encoding DNAs are placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney 293 cells (esp. the Expi293T expression system), or myeloma cells (including murine myeloma cells that do not otherwise produce immunoglobulin proteins), to obtain the synthesis of nanobodies or peptides in the recombinant host cells. Alternatively or additionally, the relatively short protein sequences of the nanobodies and peptides building blocks of this disclosure may be synthesized using solid state peptide synthesis.

[0046] Both the nanobody and the peptide “building blocks” of this disclosure may be synthesized to include synthetic or non-naturally occurring amino acids that are not part of the 20 amino acids typically used by living cells to build proteins. Examples include citrulline, p-benzoyl- phenylalanine, D-amino acids, homo amino acids, beta-homo amino acids, N-methyl amino acids, and other amino acids.

[0047] Both the nanobody and the peptide “building blocks” of this disclosure may be synthesized to include amino acids in the D-stereoisomeric amino acid confirmation. Such non-naturally occurring amino acids and / or D-stereoisomeric amino acids may impart desirable immunological and / or pharmacokinetic properties to the polypeptide constructs of this disclosure.Linkers

[0048] In the polypeptide constructs disclosed herein, the peptide is conjugated to the nanobody (e.g., a VHH), in some embodiments covalently attached. Methods of covalently conjugating two peptides (such as the nanobody and peptide within the polypeptide constructs of this disclosure) are known in the art. For example, two peptides may be fused via genetic engineering, by expressing an amino acid, peptide, or protein linking sequence between the two peptides. Examples of single amino acids or peptide sequences expressed to link peptides and nanobodies in the constructs of this disclosure, include, but are not limited to: a single glycine residue (G); glycine-glycine (GG); glycine-glycine-glycine (GGG); repeating glutamate and / or alanine, and / or lysine, residues, or combinations thereof (i.e., EAAAKEAAAKEAAAK (SEQ ID NO 17)); repeating glycine and / or serine residues, or combinations thereof (i.e., GGGGSGGGGSGGGGS (SEQ ID NO18)). A polypeptide may also be conjugated to a peptide or a non-peptide molecule via chemical crosslinking. In some embodiments, reactive chemical groups (e.g., click chemistry handles) may be incorporated into the nanobody and / or the peptide for chemical conjugation of the two molecules.

[0049] Click chemistry is a chemical approach introduced in 2001 that describes chemistry tailored to generate substances quickly and reliably by joining small units together (Angewandte Chemie International Edition (2001) 40: 2004-2021; Australian Journal of Chemistry (2007) 60: 384-395). Exemplary coupling reactions useful in linking the nanobody and the peptide of the polypeptide constructs of this disclosure include, but are not limited to, formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g, such as nucleophilic displacement of a halide or ring opening of strained ring systems); azide-alkyne Huisgon cycloaddition; thiolyne addition; imine formation; and Michael additions (e.g., mal eimide addition). Nonlimiting examples of a click chemistry handle include an alkyne handle (an unsaturated hydrocarbon containing at least one carbon-carbon triple bond), an aziridine handle, or an azide handle. The simplest acyclic alkynes with only one triple bond and no other functional groups form a homologous series with the general chemical formula CnH2n-2. Like other hydrocarbons, alkynes are generally hydrophobic but tend to be more reactive. Aziridines are organic compounds containing the aziridine functional group, a three-memberedheterocycle with one amine group (-NH-) and two methylene bridges (-CH2-). The parent compound is aziridine (or ethylene imine), with molecular formula C2H5N. Azide is the anion with the formula N3 . It is the conjugate base of hydrazoic acid (HN3). N3 is a linear anion that is isoelectronic with CO2, NCO , N2O, NC>2+and NCF. An azide can be described by several resonance structures, including -N=N+=N-

[0050] Other non-limiting, exemplary reactive groups that may be used in linking the first and peptides of the polypeptide constructs of this disclosure include, but are not limited to: acetals, ketals, hemiacetals, and hemiketals, carboxylic acids, strong non-oxidizing acids, strong oxidizing acids, weak acids, acrylates and acrylic acids, acyl halides, sulfonyl halides, chloroformates, alcohols and polyols, aldehydes, alkynes with or without acetylenic hydrogen amides and imides, amines, aromatic, amines, phosphines, pyridines, anhydrides, aryl halides, azo, diazo, azido, hydrazine, azide, and triazole compounds, strong bases, weak bases, carbamates, carbonate salts, chlorosilanes, conjugated dienes, cyanides, inorganic, diazonium salts, epoxides, esters, sulfate esters, phosphate esters, thiophosphate esters borate esters, ethers, soluble fluoride salts, fluorinated organic compounds, halogenated organic compounds, halogenating agents, aliphatic saturated hydrocarbons, aliphatic unsaturated hydrocarbons, hydrocarbons, aromatic, insufficient information for classification, isocyanates and isothiocyanates, ketones, metal hydrides, metal alkyls, metal aryls, and silanes, alkali metals, nitrate and nitrite compounds, inorganic, nitrides, phosphides, carbides, and silicides, nitriles, nitro, nitroso, nitrate, nitrite compounds, organic, non-redox-active inorganic compounds, organometallics, oximes, peroxides, organic, phenolic salts, phenols and cresols, polymerizable compounds, quaternary ammonium and phosphonium salts, strong reducing agents, weak reducing agents, acidic salts, basic salts, siloxanes, inorganic sulfides, organic sulfides, sulfite and thiosulfate salts, sulfonates, phosphonates, organic thiophosphonates, thiocarbamate esters and salts, and dithiocarbamate esters and salts. In these linkers, the reactive group may be a carboxylic acid group. These linkers may also incorporate a molecule or compound that imparts additional benefits to the polypeptide construct. For example, these linkers may incorporate one or more polyethylene glycol (PEG) molecules, which may impart a longer half-life to the polypeptide construct following administration to a subject. Examples of such additional linkers include:GGSGGGSGGSGGGS (GlySer linker 2x; SEQ ID NO: 16)

[0051] In establishing covalent links between the nanobody and the peptides of this disclosure, the C-terminus of the nanobody may be fused to the N-terminus of the peptide. Alternatively, the N-terminus of the nanobody may be fused to the C-terminus of the peptide.Alternatively, the fusion inhibitor peptide may be fused at the C-terminus to the C-terminus of the nanobody.

[0052] Another useful link between the nanobody and the fusion inhibitor peptide in the peptide constructs of this disclosure may be created by a sortase-mediated peptide ligation processes (described, e.g., in U.S. Patent No. 9,751,945; and International Patent Applications PCT / US2010 / 000274, and PCT / US2011 / 033303). As described in detail therein, sortases are enzymes found in Gram-positive bacteria that catalyze a transpeptidation reaction by cleaving a surface protein substrate at the cell wall and anchoring the protein to the bacterial cell wall. Sortase A, the most widely studied class of sortases, binds to a specific peptide motif, cleaves between specific amino acids, and forms an acyl-enzyme intermediate. Sortase A is a valuable tool in protein and peptide chemistry that is frequently used in bioconjugation and semisynthesis of proteins. The process of synthesizing protein and peptide constructs using sortases typically involves: 1) recognition and cleavage: the sortase recognizes a specific peptide motif at the C-terminal end of the target protein and cleaves between specific amino acids (typically threonine and glycine); 2) transpeptidation: after cleavage, the sortase forms an acyl-enzyme intermediate between its active site cysteine thiol and the carboxyl group of the threonine residue; and 3) anchoring: the acyl-enzyme intermediate undergoes nucleophilic attack of the peptidoglycan crossbridge amino group, allowing for site-specific and covalent attachment of the target protein or peptide to another protein or peptide molecule. The sortase transacylation reaction provides means for efficiently linking an acyl donor with a nucleophilic acyl acceptor. This principle is widely applicable to many acyl donors and a multitude of different acyl acceptors, and the sortase reaction has been widely employed in the synthesis of biomolecules such as ligating proteins and / or peptides to one another, ligating synthetic peptides to recombinant proteins, linking reporting molecules to a protein or peptide, joining a nucleic acid to a protein or peptide, conjugating a protein or peptide to a solid support or polymer, and linking a protein or peptide to a label.

[0053] The transpeptidation reactions catalyzed by sortases result in the ligation of species containing a transamidase recognition motif with those bearing one or more N-terminal glycine residues. The sortase recognition motif may be a sortase recognition motif such as LPXT or LPXTG (using the single letter amino acid symbols; L = Leucine; P = Proline; X = any amino acid (natural or non-natural); T = Threonine). As is known in the art, the substitution of the C-terminal residue of the recognition sequence with a moiety exhibitingpoor nucleophilicity once released from the sortase provides for a more efficient ligation. In these synthesis reactions, the sortase may be Sortase A (SrtA), but it should be noted that any sortase or transamidase catalyzing a transacylation reaction can be used in establishing linkers between the nanobody and peptides of the polypeptide constructs of this disclosure.

[0054] In linking the nanobodies and peptides of the polypeptide constructs of this disclosure, sortase-catalyzed transacylation reactions may be used to link click chemistry handles onto the C- or N-terminal amino acids of the nanobody and / or the peptide. These click chemistry handles are then quickly and efficiently combined to form a covalent linker between the nanobody and peptide.

[0055] In examples of such linking strategy, the nanobody of this disclosure may be synthesized with an amino acid sequence recognized by a transamidase enzyme at the C-terminus of the nanobody. This nanobody is contacted with the peptide of this disclosure comprising a nucleophilic acyl acceptor containing a NH2 moiety at the N-terminus of the peptide, in the presence of a transamidase enzyme to create a linker between the nanobody and peptide; the linker comprising the transamidase recognition sequence and the nucleophilic acyl acceptor, thereby establishing a covalent linker between the C-terminus of the nanobody and the N- terminus of the peptide. In a specific example illustrated in FIG. 3 of this disclosure, the nanobody of this disclosure may be synthesized with a C-terminus amino acid sequence comprising the LPXT or LPXTG motifs recognized by Sortase A. This construct, comprising a nanobody linked to a sortase recognition sequence linked to a His-tag, may be efficiently formed by recombinant expression and then purified by binding and then removing the His tag. This peptide is contacted with the peptide of this disclosure comprising between one and five glycine amino acid residues, and optionally one or more histidine residues, in the presence of Sortase A to create a linker between the nanobody and peptides of this disclosure; the linker comprising the LPXT(G)i-s amino acid sequence, wherein X may be E (glutamic acid), D (aspartic acid), A (alanine), N (asparagine), Q (glutamine), K (lysine), or R (arginine).

[0056] In another example of such linking strategies, a covalent linker is established between the C- terminus of the nanobody and the C-terminus of the peptide. In the specific example illustrated in FIG. 4 of this disclosure, the nanobody of this disclosure may be synthesized and purified by recombinant expression with a C-terminus amino acid sequence comprisingthe LPXT or LPXTG motifs recognized by Sortase A, as described above. This peptide is contacted with a short peptide comprising one to five glycine residues conjugated at the C- terminus to an azide in the presence of Sortase A to create a linker between the nanobody and the azide; the linker comprising the LPXT(G)i-s amino acid sequence, wherein X may be E, D, A, N, Q, K, or R, as described above. The peptide is linked to the click chemistry handle dibenzocyclooctyne group (DBCO). DBCO may be attached to a cysteine amino acid residue in the peptide (e.g., the Cys added at the C-terminus of a peptide of this disclosure) using mal eimide (Mai), and optionally with an intervening linker of PEG3 (DBC0-PEG3-Mal, Catalog # CP-2030, Conju-Probe, LLC, California). The DBCO-Mal linker reacts with the azide established at the C-terminus of the nanobody to form an LPET- (glycine)i-5-triazole linker between the C-terminus of the nanobody and the C-terminus of the peptide. Conjugation carried out using DBC0-PEG3-Mal click chemistry handle on the peptide results in a PEG linker between the first and the peptides.

[0057] The click chemistry handle may be incorporated into the nanobody and / or the fusion inhibitor peptide of this disclosure in any manner and at any position that can be envisioned by those of skill in the art. For example, the click chemistry handle attached to the peptide of this disclosure may be attached to a central amino acid, or to the side chain of any amino acid, or to an amino acid of the C-terminus of the peptide, or to an amino acid of the N- terminus of the peptide, or any other position in the peptide.Polypeptide constructs

[0058] It will be appreciated that the order of the first building block and the second building block in the polypeptide (orientation) may be chosen according to the needs or goals of the person skilled in the art preparing the construct, as well as the relative affinities which may depend on the location of these building blocks in the polypeptide. Whether the polypeptide comprises a linker, is a matter of design choice. However, some orientations, with or without linkers, may provide preferred binding characteristics in comparison to other orientations. For instance, the order of the first and the second building block in the polypeptide of the invention can be (N-terminus to C-terminus, nanobody-[linker]-peptide) or (C-terminus to C-terminus, nanobody-[linker]-peptide). All orientations are encompassed within the polypeptide constructs of this disclosure. Polypeptide constructs of this disclosurethat contain an orientation of building blocks that provides desired (binding and / or HIV- neutralizing) characteristics can be easily identified by routine screening, for instance as exemplified in Example 1, infra.

[0059] Exemplary polypeptide constructs of this disclosure comprising a nanobody linked to a peptide include, but are not limited to:Nb-3F11 (SEQ ID NO:4) - Linker - C34 (SEQ ID NO: 11) Nb-281E10 (SEQ ID NO: 5) - Linker - C34 (SEQ ID NO: 11) Nb-281F12 (SEQ ID NO:6) - Linker - C34 (SEQ ID NO: 11) Nb-CD4-bl (SEQ ID NO:8) - Linker - C34 (SEQ ID NO:11) Nb-J3 (SEQ ID NO: 1) - Linker - C34 (SEQ ID NO: 11) Nb-30 (SEQ ID NO:7) - Linker - C34 (SEQ ID NO: 11), and Nb-2E7 (SEQ ID NO:3) - Linker - C34 (SEQ ID NO: 11).

[0060] As noted above, the polypeptide constructs of this disclosure may be produced by expression of the peptide sequences of the nanobody and peptide constructs, with or without an intervening amino acid or peptide linker. Exemplary polypeptide constructs of this disclosure comprising a nanobody linked to a fusion inhibitor peptide, which are produced by expression as a single peptide include, but are not limited to: 3F11-C34 (SEQ ID NO: 19) C34-3F11 (SEQ ID NO:20) 3F11-C34N10Q (SEQ ID N0:21) 3F11-RIGID-C34 (SEQ ID NO:22) 3F11-FLEX-C34 (SEQ ID NO 23) Pharmaceutical Compositions

[0061] For therapeutic use, pharmaceutical compositions comprising one or more of the polypeptide constructs of this disclosure may be prepared by mixing a polypeptide construct with one or more pharmaceutically acceptable carriers. As used herein, “pharmaceutically acceptable carrier” means buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. The carrier(s) should be “acceptable” in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient. Useful formulations canbe prepared by methods well known in the pharmaceutical arts. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Pharmaceutically acceptable carriers may include solvents, dispersion media, isotonic agents, and the like. The carrier may be liquid, semi-solid (e.g., pastes), or solid carriers. Examples of carriers include water, saline solutions or other buffers (such as phosphate, citrate buffers), oil, alcohol, proteins (such as serum albumin, gelatin), carbohydrates (such as monosaccharides, di saccharides, and other carbohydrates including glucose, sucrose, trehalose, mannose, mannitol, sorbitol or dextrins), gel, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, stabilizers, preservatives, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA; salt forming counter-ions such as sodium; non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG), and combinations thereof.

[0062] These pharmaceutical formulations may contain more than one active compound, e.g., one or more polypeptide constructs, in combination with one or more additional beneficial compounds for preventing and treating HIV infections.

[0063] The active ingredients can be combined with the carrier in any convenient and practical manner, e.g., by admixture, solution, suspension, emulsification, encapsulation, absorption, and the like, and can be made in formulations such as tablets, capsules, powders (including lyophilized powders), syrups, suspensions including suspensions suitable for injections, infusions, or the like. Sustained-, extended- and / or controlled-release preparations may also be prepared.

[0064] Pharmaceutical compositions containing polypeptide constructs of this disclosure may be presented in a unit dosage form and may be prepared by any suitable method. A pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes intended for therapeutic administration of the polypeptide constructs of this disclosure are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, and rectal administration. Exemplary routes of administration for polypeptide constructs of this disclosure are parenteral administration and topical administration.

[0065] These pharmaceutical formulations preferably are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition islyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.Methods of Treatment and Prevention

[0066] The polypeptides of this disclosure inhibit binding and viral entry of HIV into CD4+ T-cells by binding to 1) T-cell surface proteins that participate in HIV recognition, binding, and entry (including the CD4 receptor and / or the CXCR4 coreceptor), and / or 2) the HIV-1 Env protein complex (including gp!20 and / or gp41). By binding these proteins, the polypeptide constructs of this disclosure prevent HIV from entering the T-cell. HIV cannot survive for prolonged periods of time outside of a cellular environment. Thus, if HIV cannot enter the cell and remains in the extracellular environment, HIV will be disposed of by the body, resulting in the lower HIV-titer in an infected subject.

[0067] Methods for determining the amount of HIV in a subject (the HIV-titer) are known and routinely used in treating and studying HIV infection. Briefly, a blood sample from a subject is provided and the amount of HIV (e.g, the number of HIV-particles) is determined either directly (by assaying for the presence of HIV) or indirectly (e.g., by assaying for the presence of antibodies against HIV). Determining the presence of HIV, or antibodies against HIV, can be performed, for example, by ELISA. Additional methods of determining the amount of HIV in a subject include functional inhibition assays, assays to determine the presence and amount of specific antigens such as the p24 antigen test (commercially available through PerkinElmer and Advanced Bioscience Laboratories), and assays to determine the presence and amount of specific nucleic acids encoding the HIV genome, through reverse transcriptase activity (e.g., ExaVir Load; Cavidi Tech-AB, Uppsala, Sweden).

[0068] The methods of treatment and prevention of this disclosure are applicable to any form of HIV including HIV- 1 and HIV-2, and all the subclasses, e.g., HIV-1B, HIV-1D, etc. These methods may also be applicable to viruses related to HIV, such as the simian virus SIV.

[0069] As used herein, a subject includes a mammal that is susceptible to HIV infection (e.g., a human), or infection by a related virus such as SIV (e.g., a monkey). The subject may be a human or a primate. The subject may be receiving, or has received, one or more anti-HIV treatment or prevention regimens (e.g, anti-retroviral therapy, ART, or PREP or acomponent thereof). The subject may be infected with HIV that has become resistant to at least one other anti -HIV agent. Subjects include humans or monkeys that are at risk for HIV-related diseases or disorders including patients who have come in contact with an infected person or who have been exposed to HIV in some other way. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of HIV- related disease or disorder, such that a disease or disorder is prevented or, alternatively, delayed in its progression.

[0070] This disclosure provides polypeptide constructs as described above, for use in treating a subject in need of treatment, for example, a subject infected with HIV, including specifically, HIV-1.

[0071] This disclosure also provides a method for delivering a prophylactic and / or therapeutic polypeptide to a specific location, tissue or cell type in the body, the method comprising the steps of administering to a subject at least one polypeptide of this disclosure, or a pharmaceutical composition comprising the same.

[0072] This disclosure also provides methods for treating a subject in need thereof comprising administering at least one polypeptide of this disclosure to the subject.

[0073] In these methods, the subject may be infected with HIV R5, HIV X4, and / or HIV X4R5.

[0074] In one aspect, the disclosure provides a method for treating a subject infected with HIV, the method comprising administering to the subject at least one polypeptide construct of this disclosure comprising a nanobody linked to a peptide to treat the infection. As described above, the polypeptide constructs may function to inhibit or completely block viral entry of HIV into T-cells. In these methods, the administered polypeptide construct may have only minimal undesirable side effects.

[0075] As used herein, the phrase “treating a subject infected with HIV” refers to any method that results in a reduction in the severity of symptoms (e.g, opportunistic microbial infections) or number of symptoms (e.g, number of opportunistic infections) associated with HIV infection in a subject, or reduction in the HIV titer in the subject.

[0076] In these methods, treating a subject infected with HIV may result in an increase in the white blood cell count in the treated subject. Similarly, in these methods, treating a subject infected with HIV may result in an increase in the number of CD4+ T-cells in the subject.Similarly, in these methods, treating a subject infected with HIV may result in a decrease inthe HIV-titer in the treated subject. Accordingly, this disclosure provides methods for lowering the HIV-titer in a subject, the method comprising administering to the subject a therapeutically effective amount of a polypeptide construct of this disclosure to lower the HIV-titer in the subject. In these methods, the treatment may also result in a decrease in the number of opportunistic microbial infections in the treated subject.

[0077] Whether treatment administered in these methods is effective can be assessed, for instance, by determining the change in one or more physiological parameters associated with HIV infection (e.g, lowering of HIV-titer, decrease in the number of infected cells, increase in the amount of CD4+ T-cells), or by assessing the whole body health of the treated subject (e.g., decrease in the number of opportunistic infections).

[0078] Anti -HIV treatment regimens (e.g., ART or PREP), including the administration ofHIV- protease inhibitors and HIV-reverse transcriptase inhibitors, are associated with a number of unwanted side effects, including liver toxicity, bloating, diarrhea, fatigue, headache, nausea, loss of appetite, etc. The compositions and methods provided in this disclosure for treating a subject infected with HIV may induce minimal unwanted side effects.

[0079] In these methods of treatment and prevention, the administration of the polypeptide constructs of this disclosure may delay or prevent the development of resistance to the treatment for at least 3 months, or at least 6 months, or even longer, such as 9 months, 11 months, 1 year, 1.5 years, 2 years, or even longer.

[0080] In these methods, the polypeptide constructs of this disclosure and / or the pharmaceutical compositions comprising the same can be administered in any suitable manner, depending on the specific pharmaceutical formulation to be used. Thus, the polypeptides of this disclosure and / or the pharmaceutical compositions comprising the same can be administered orally, intraperitoneally (e.g., intravenously, subcutaneously, intramuscularly, or via any other route of administration that circumvents the gastrointestinal tract), rectally, vaginally, intranasally, transdermally, topically, by means of a suppository, or by inhalation. The clinician or the subject will be able to select a suitable route of administration and a suitable pharmaceutical formulation or composition to be used in such administration, depending on the disease or disorder to be prevented or treated and other factors known to the clinician and the subject.

[0081] In these methods, the polypeptides of this disclosure and / or the pharmaceutical compositions comprising the same are administered according to a regime of treatment that is suitable for preventing and / or treating an HIV infection. The clinician or the subject will be able to determine a suitable treatment regimen, depending on factors such as the stage of the HIV infection to be treated, the severity of the HIV infection to be treated and / or the severity of the symptoms thereof, the specific route of administration and pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician or the subject. Generally, the administration regimen will comprise administration of one or more polypeptides of this disclosure, or one or more pharmaceutical compositions comprising the same, in one or more therapeutically effective amounts or doses. The specific amount(s) or doses to be administered may be determined by the clinician or the subject.

[0082] In these methods, the polypeptides of this disclosure may also be used in combination with one or more additional HIV therapies or preventative treatments, i.e., as a combined treatment regimen, which may or may not lead to a synergistic effect. This may include the use of the polypeptides of this disclosure in combination with other pharmaceutically active compounds that are or can be used for the prevention and / or treatment of an HIV infection and / or any opportunistic infection, disease and / or disorder associated with or resulting from HIV infection. As a result of such combined therapy, a synergistic effect may or may not be obtained. When additional compounds are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition.

[0083] To evade HIV resistance and prolong efficacy, contemporary anti-HIV treatment regimens comprise a cocktail of anti-HIV drugs. Hence, it may be advantageous to include one or more polypeptides of this disclosure in an anti-HIV treatment regimen, such as ART therapy or a component thereof. In some embodiments, the subject may be treated with a polypeptide of this disclosure and e.g. one or more protease inhibitors such as amprenavir (AMP), atazanavir (ATV), indinavir (IDV), lopinavir (LPV), nelfmavir (NFV), ritonavir (RTV) or saquinavir (SQV); and / or reverse transcriptase inhibitors (RTIs) such as a nonnucleoside reverse transcriptase inhibitor (NNRTI) [abacavir (ABC), delavirdine (DLV), efavirenz (EFV), nevirapine (NVP) and tenofovir (TFV)]; or a nucleoside analogue reversetranscriptase inhibitor (NRTI) [didanosine (ddl), stavudine (d4T), lamivudine (3TC) and zidovudine (ZDV)].

[0084] This disclosure also provides methods of suppressing or preventing infection of a T-cell expressing CD4 and / or CXCR4 by an HIV virus, comprising contacting the T-cell with one or more of the polypeptide constructs of this disclosure to suppress or prevent infection of the T-cell by the virus. This method may suppress infection of a T-cell in vitro or in vivo.

[0085] This disclosure also provides methods of inhibiting binding of HIV to T-cell surface proteins, including the CD4 receptor and / or the CXCR4 coreceptor. This method comprises contacting the T-cell surface protein with at least one polypeptide construct of this disclosure to inhibit binding of the virus to the T-cell surface protein, wherein contacting the T-cell surface protein with the polypeptide construct inhibits binding of HIV to the T-cell surface protein. In these methods, contacting the T-cell surface protein, such as the CD4 receptor and / or the CXCR4 coreceptor, with the polypeptide construct does not displace a natural ligand from the T-cell surface protein.

[0086] This disclosure also provides methods of inhibiting the onset or progression of an HIV infection or an HIV-associated disorder in a human subject by inhibiting fusion of an HIV to CXCR4+ and / or CD4+ immune cells in the subject, comprising administering to the subject a fusion-inhibitory dose of a polypeptide of this disclosure. In these methods, the HIV may be resistant to (i) one or more HIV protease inhibitors, (ii) one or more HIV reverse transcriptase inhibitors, and / or (iii) one or more HIV protease inhibitors and one or more HIV reverse transcriptase inhibitors. In these methods, the administration of the polypeptide inhibits the onset or progression of the HIV infection or the HIV-associated disorder in the subject.

[0087] This disclosure also provides a method of preventing HIV infection in a subject, the method comprising administering to the subject at least one polypeptide construct of this disclosure to prevent an HIV infection in the subject. In these methods, the administered polypeptide construct s) may inhibit binding of HIV to a T-cell surface protein such as the CD4 receptor and / or the CXCR4 coreceptor. In these methods, the administered polypeptide construct(s) may not displace a natural ligand from the T-cell surface protein. Thus, these methods include preventing HIV infection in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one polypeptide construct of thisdisclosure to prevent infection of the subject by HIV. In these methods, the HIV infection may be prevented by precluding HIV from entering and / or accumulating in CD4+ T-cells in the subject. Thus, the HIV infection may be prevented even after a subject has been exposed to an HIV, and may have one or more signs of having been exposed to an HIV, by preventing HIV from entering and / or accumulating in the CD4+ T-cells in the subject.

[0088] In these methods, preventing an HIV infection refers both to complete and partial prevention (e.g, a percentage reduction, for example about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or higher or lower or intermediate percentages of getting infected by HIV). For example, a subject may have a 50% chance of getting infected by HIV upon exposure to HIV through a specific route (e.g., intravenous injection), but administration of one or more polypeptide constructs of this disclosure may reduce the chance of infection to only a 10% chance of infection upon exposure (thus resulting in an 80% reduction in the chance of infection following exposure).

[0089] Prevention of infection can be determined using established simian models of HIV and SIV infection. For instance, a group of monkeys may be administered one or more of the polypeptides constructs of this disclosure, and subsequently be exposed to HIV / SIV, while a control group, which is also exposed to HIV / SIV, is not administered the polypeptide construct s). If the incidence of HIV / SIV infection in the group to which the polypeptide constructs have been administered is lower than in the control group, then the polypeptide constructs are effective in preventing infection by HIV.

[0090] This disclosure also provides methods of reducing the likelihood of a human subject contracting an HIV infection, by administering to the subject one or more polypeptide constructs of this disclosure, to thereby reduce the likelihood of the subject’s contracting an HIV infection at a time after the administration of the polypeptide construct(s).

[0091] This disclosure also provides methods of reducing an increase in HIV virus titer, HIV viral replication, HIV virus proliferation, or an amount of an HIV viral protein in a subject. These methods include administering to the subject an amount of one or more polypeptide constructs of this disclosure effective to reduce an increase in HIV titer, HIV viral replication, and / or an HIV protein of one or more HIV strains or isolates in the subject.

[0092] This disclosure also provides methods of reducing HIV viral replication or spread of HIV infection to additional host cells or tissues by contacting a mammalian cell with one or more polypeptide constructs of this disclosure.

[0093] In any of these methods of treating or preventing or reducing the likelihood of an HIV infection, the one or more polypeptide construct(s) of this disclosure are preferably administered within a pharmaceutical formulation.EXAMPLES

[0094] The compositions and methods of this disclosure are further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, and published patent applications as cited throughout this application) are hereby expressly incorporated by reference. Example 1 : Making and Analyzing Polypeptide Constructs of this Disclosure

[0095] Nanobodies were produced using recombinant expression in BL21(DE3) E. coli using a pet26b(+) expression vector, and purified using NiNTA affinity chromatography as described previously (Cabalteja, etal., (2022) ACS Chem. Biol. 17:2296-303). The peptides used to build the polypeptide constructs of this disclosure were prepared by standard solid phase peptide synthesis using fluorenylmethyloxy carbonyl (Fmoc)-amino acid residues to protect the polypeptide backbone amines (see, e.g., Cameron, L., etal., J. Chem. Soc., Chem. Commun., 1987, 270-272). Each of the synthesized peptides was removed from the resin and deprotected, dissolved in water, and purified with reverse-phase preparative HPLC. Purity and identity of the synthesized nanobodies and peptides was confirmed mass spectrometry and SDS-PAGE gel. FIG. 1 shows an example of a stained SDS-PAGE gel run with seven of the synthesized nanobodies of this disclosure.

[0096] The protein binding specificity of the individual nanobodies was confirmed using fluorescence-activated cell sorting (FACS) with different fluorophores and two cell lines (Sarzotti-Kelsoe, et al., 2014 J. Immunol. Methods. 409: 131-46). The TZM-bl cell line is derived from a HeLa cell clone that was engineered to express CD4, CCR5, and CXCR4. The HEK-293T cells express MHC-1 and CXCR-4 and do not express the CD4 receptor. FIGS. 2A and 2B show the FACS counts demonstrating that, as expected, the CD4-targeting nanobody Nb-3F11 (SEQ ID NO:4) binds strongly to TZM-bl cells (FIG. 2A) but not theHEK293 cells (FIG. 2B). Additionally, the CXCR4-targeting nanobodies Nb-281E10 (SEQ ID NO:5) and Nb-281F12 (SEQ ID NO:6) correctly bind to both cell lines (FIGS. 2A and 2B) that endogenously express CXCR4. The positive control (Nb-30; SEQ ID NO:4) correctly binds the HEK293 cells expressing the MHC-1 protein (FIG. 2B), and a negative control (Nb-6E) did not bind to either cell line. This confirmed that the synthesized nanobodies have the expected cell surface protein binding specificity.

[0097] The protein binding sensitivity of the individual nanobodies was confirmed using the same FACS assay with TZM-bl cells expressing the CD4 receptor at concentrations as low as 0.1 nM. FIGS. 3A and 3B show the FACS counts for the negative control (Nb-6E) and the positive control (MHC-1 binding Nb-30) and for the CD4-targeting nanobodies Nb-3F11 (SEQ ID NO:4; FIG. 3 A) and Nb-CD4-bl (SEQ ID NO:8; FIG. 3B) indicating detectable binding to TZM-bl cells expressing CD4 at concentrations from lOOnM down to O.lnM. These data confirm the synthesized nanobodies have very good protein binding sensitivity.

[0098] The epitope binding specificities of the CD4 receptor-binding Nb-3F11 (SEQ ID NO:4) and Nb-CD4bl(SEQ ID NO: 8) nanobodies were further characterized in binding competition assays to determine whether these two nanobodies bind to the same or separate epitopes on the CD4 receptor. Using the TZM-bl cells expressing CD4 at concentrations from lOOnM down to O.lnM, binding competition assays were conducted using both a labeled (with biotin-avidin label) and unlabeled copy of each of the Nb-3F11 and Nb-CD4bl nanobodies. The results of these binding competition assays are quantified in the graph of FIG. 4. The self-competition control assays were conducted between a labeled and an unlabeled copy of each nanobody (labeled CD4B1-CD4B1BA and 3Fl l-3Fl lBA in FIG. 4) and, as expected, showed competition for binding to the TZM-bl cells expressing the four different concentrations of the CD4 receptor. Alternatively, the binding competition assays conducted between the two nanobodies (labeled CD4bl-3Fl 1BA and 3F1 l-CD4blBA in FIG. 4) showed no competition for binding to the TZM-bl cells expressing the four different concentrations of the CD4 receptor, providing clear evidence of different binding sites on the CD4 receptor for the Nb-3F11 and Nb-CD4bl nanobodies.

[0099] Polypeptide constructs of this disclosure were prepared by linking a nanobody to an HIV fusion inhibitor (FI) peptide resulting in nanobody-linker-peptide constructs. As describedabove, and depicted in FIG. 5, the nanobodies used in these constructs, which bind to an epitope on either one of:1) an extracellular portion of the HIV Env protein; or2) a cell surface receptor on CD4+ T-cells; were linked to the peptide by expressing the nanobody amino acid sequence with a Sortase A recognition sequenceSortagging) at the C-terminus, as well as a (His)e tag (for purification after expression; SEQ ID NO: 14). The FI peptide was expressed with at least three glycine amino acid residues at the N-terminus of the peptide (for example: G3C34Cys; SEQ ID NOTO). The sortagged-nanobody and the (Gly)j-N terminal FI peptide were linked to form a polypeptide construct comprising: the nanobody-linker-peptide sequence, wherein the linker has the amino acid sequence: -LPETGGG- (SEQ ID NO: 13) and the nanobody and the peptide are bound in a C-terminus to N-terminus orientation (nanobody to peptide, respectively) by combining them in the presence of 5M Sortase A.

[0100] As described above, and depicted in FIG. 6, the same nanobodies used in these constructs were also bound to these FI peptides in a C-terminus to C-terminus orientation (nanobody to peptide, respectively). To prepare these C-C terminus polypeptide constructs, the nanobody amino acid sequence was expressed and purified with a Sortase A recognition sequence (z.e., Sortagging) at the C-terminus, as well as a (His)e tag (for purification after expression; SEQ ID NO: 14). An azide was attached to the C-terminus of this nanobody by incubating with a 3 -glycine amino acid peptide covalently linked to the azide in the presence of 5M Sortase A to form a nanobody-linker-azide construct, wherein the linker is LPETGGG (SEQ ID NO: 13). The FI peptide was prepared for linking with this nanobody-linker-azide construct by expressing the FI peptide with a C-terminal cysteine (Cys) amino acid (for example: C34Cys; SEQ ID NO:11), which is linked to the dibenzocyclooctyne-maleimide (DBCO- Mal) click chemistry handle to form a FI peptide-Cys-DBCO-Mal construct. The nanobody- linker-azide construct was fused with the FI peptide-cysteine-DBCO construct to form a nanobody-linker-peptide construct, wherein the linker is LPETG3 -tri azole, and the nanobody and the peptide are bound in a C-terminus to C-terminus orientation (nanobody to peptide, respectively).

[0101] Following the synthesis of these polypeptide constructs, comprising nanobodies linked to FI peptides, the finished polypeptides were isolated and purified. FIG. 7 shows an example of astained SDS-PAGE gel run with six of the synthesized polypeptide constructs of this disclosure demonstrating the purity of the synthesized polypeptide constructs. In FIG. 7, the FI peptide C34 was linked with the nanobodies: Nb-J3 (SEQ ID NO: 1); Nb-2E7 (SEQ ID NO:3); Nb-3F11 (SEQ ID NO:4); Nb-281F12 (SEQ ID NO:6); Nb-281E10 (SEQ ID NO:5); and Nb-30 (SEQ ID NO:7), appearing left to right in FIG. 7.Example 2: HIV Binding Inhibition by Polypeptide Constructs of this Disclosure

[0102] The efficacy of polypeptide constructs of this disclosure to block HIV-1 pseudovirus infection was measured using the TZM-bl assay for standardized assessments of neutralizing proteins against HIV-1 (Sarzotti-Kelsoe, etal., 2014. Journal of Immunological Methods 409: 131-146); Montefiori, D.C., 2009. In: Vinayaka, G.V.K., Prasad, R. (Eds.), HIV Protocols, Second edition. Methods Mol. Virol, 485:395-405). The assay measures neutralization as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses.

[0103] The pseudovirus neutralization assay was used to compare the neutralization potencies of individual nanobodies, fusion inhibitors, and nanobody-fusion inhibitor constructs of this disclosure (as previously described in Montefiori, 2005, Current protocols in immunology, Edited by John E. Coligan et al., Chapter 12, Unit 12.11). Briefly, pseudoviruses were generated in HEK293T cells by co-transfection of an Env-expressing vector and a replication-incompetent backbone plasmid. Neutralization was assessed by measuring the reduction in luciferase reporter gene expression in the presence of any one of the tested inhibitor proteins following a single round of pseudovirus infection in TZM-bl cells engineered to express CD4 and CCR5. Nanobodies or fusion inhibitor peptides or nanobody-peptide constructs were pre-incubated with infectious viral units of HIV- 1 strain YU2 in a dilution series at 37°C before adding TZM-bl cells for a two-day incubation. Cells were then lysed and luciferase expression was measured. Nonlinear regression analysis was used to calculate the concentrations at which half-maximal inhibition was observed (IC50 values) as described in Klein et al., 2009, PNAS, 106:7385-90.

[0104] FIGS. 8A-8D show neutralization curves for nanobodies, fusion inhibitor peptides, and polypeptide conjugates of this disclosure against the YU2 strain of HIV-1 pseudoviruses. FIG. 8A shows the neutralization curve for the C34 fusion inhibitor peptide (G3-C34-Cys; SEQ ID NO: 10), which binds with the N-terminal heptad repeat of the gp41 Env protein,and four nanobody constructs of this disclosure comprising a nanobody bound to the C34 fusion inhibitor peptide. Each of these polypeptide constructs comprised a nanobody covalently linked to the C34 peptide through a LPETGGG (SEQ ID NO: 13) linker between the C-terminus of the nanobody and the N-terminus of the C3 peptide (i.e., Nanobody- LPETG3-C34). The nanobodies in these constructs and their IC50 values are provided in Table 2.

[0105] FIG. 8B shows another neutralization curve created from a second HIV pseudovirus neutralization assay that used the same constructs described above for the first HIV pseudovirus neutralization assay depicted in FIG. 8A, with the exception that nanobody 281E10 was replaced by 28 IF 12. The nanobodies in these constructs and their IC50 values are provided in Table 3:

[0106] These first two HIV pseudovirus neutralization assays (FIGS. 8 A and 8B) indicate that the Nanobody -peptide conjugates of this disclosure that target the CD4 T-cell surface proteins, CD4 receptor and CXCR4 coreceptor) are 500-fold more potent than the C34 fusion inhibitor peptide alone.

[0107] FIG. 8C shows another neutralization curve created from a third HIV pseudovirus neutralization assay that used two individual nanobodies [Nb-3F11 (SEQ ID NO:4) and Nb- 28 IF 12 (SEQ ID NO:6)], and polypeptide constructs comprising a nanobody covalently linked to the C34 peptide through a LPETGGG (SEQ ID NO: 13) linker between the C- terminus of the nanobody and the N-terminus of the C3 peptide (z.e., Nanobody -LPETG3- C34). The nanobodies in these polypeptide constructs were Nb-3F11 (SEQ ID NON, binds to CD4 receptor); Nb-281F12 (SEQ ID NO:6, binds to CXCR4 receptor); Nb-VHH05 (a non-binding control nanobody); Nb-CD4bl(SEQ ID NO: 8, binds to CD4 receptor). The results of this third HIV pseudovirus neutralization assay show that Nb-3F11 has weak antiviral activity by itself (without being bound to the C34 fusion inhibitor peptide); the CD4bl-C34 polypeptide construct had measurable HIV neutralization activity; the 3F11- C34 polypeptide construct showed more potent HIV neutralization activity than the CD4bl- C34 polypeptide construct; the VHH05-C34 polypeptide construct showed similar HIV neutralization activity to the C34 fusion inhibitor peptide alone, indicating that this nonbinding control nanobody did not interfere with C34 peptide binding of the gp41 Env protein; and the CXCR4-binding nanobody (Nb-281F12) tested by itself (without binding to a fusion inhibitor peptide) is inactive in this neutralization assay.

[0108] FIG. 8D shows another neutralization curve created from a fourth HIV pseudovirus neutralization assay that tested one nanobody -peptide construct (Nb-30-C34; nanobody that binds to HMC-1 protein on T-cell surface), one antiviral peptide (EP417), and four individual nanobodies: 1) Nb-3F11 (SEQ ID NON, binds to CD4 receptor); Nb-281E10 (SEQ ID NO:5, binds to CXCR4 receptor); Nb-281F12 (SEQ ID NO:6, binds to CXCR4 receptor); and Nb-CD4bl (SEQ ID NO:8, binds to CD4 receptor). The results of this fourth HIV pseudovirus neutralization assay show that the HIV neutralization activity ofNb- CD4bl looks similar to that of Nb-3F11, despite the earlier showing (Example 1, FIG. 4, supra) that these two antibodies do not bind to the same epitope on the CD4 receptor; Nb-30 nanobodies, which are general T-cell surface tethering nanobodies that do not bind to or provide co-receptor blockade, show high HIV neutralization efficacy; the EP417 (antiviral peptide) showed no activity in this assay.

[0109] The compiled IC50 values for all pseudovirus neutralization assays are shown as the mean value (in nM) in Table 4:

[0110] Table 4

[0111] Example 3 : Binding Inhibition in HIV Strains from Different Viral Clades

[0112] To supplement the efficacy testing conducted against a single strain of HIV pseudovirus, the peptide constructs of this disclosure were tested against nine additional strains of HIV pseudovirus, as well as a negative control pseudovirus. These nine additional HIV strains were chosen to come from different viral clades and have differing specificities for the coreceptors used for infection (CXCR4 vs. CCR5). HIV infection inhibitors with broad coverage against different viral strains are highly desirable for the development of broadly active anti-HIV compounds.

[0113] Four nanobody-HIV fusion inhibitor peptide constructs of this disclosure were chosen for testing across the field of nine additional strains of HIV pseudovirus, along with theindividual nanobodies (28 IF 12, CD4B1, 3F11) and HIV fusion inhibitor (G3,C34) “parental” peptides. Also included as controls were the antiviral drug (“enfuvirtide”) and the antiviral monoclonal antibody VRC01. Table 5 lists the IC50 values (in pg / ml) and Table 6 lists the number and percentage neutralized for the constructs (and individual parental peptides) tested over the ten HIV pseudoviruses (9 new and 1 from the initial testing described above) and one control (non-HIV) virus (Note that median and geometric mean titers were calculated only for samples with calculated IC50s). Table 7 lists the IC50 values listed by the coreceptor bound by the constructs tested (CCR5 vs CXCR4), and Table 8 lists the same data expressed as the IC80 value (again in pg / ml).

[0114] The efficacy of polypeptide constructs of this disclosure to block HIV-1 pseudovirus infection in these nine additional HIV strains was measured using the TZM-bl assay describe above. The pseudovirus neutralization assay described above was again used to compare the neutralization potencies of individual nanobodies, fusion inhibitors, and nanobody-fusion inhibitor constructs of this disclosure.

[0115] These data demonstrate that the constructs of this disclosure which showed the highest activities in initial assays were also very potent against all of the additional HIV strains tested. As expected, all compounds were inactive against the negative control pseudovirus. Example 4: Binding Inhibition by Constructs expressed in Mammalian Expression System

[0116] Constructs of this disclosure were prepared by expressing the full length of the construct in a mammalian recombinant expression system (HEK293 cells) as opposed to the bacterial expressed used for individual nanobodies. Five recombinantly expressed constructs were prepared and tested. Each of these five constructs were built around the combination of the 3F11 nanobody (SEQ ID NO:4) and the C34 HIV fusion inhibitor peptide (SEQ ID NOV). Variations on this construct and the peptide sequences linking these two peptides were used to test the feasibility and efficacy of the expression system and constructs created using this expression system. The five Nb-FI peptide constructs prepared and tested included:

[0117] 3F11-C34_exp (the 3F11 nanobody linked to the C34 HIV fusion inhibitor peptide through a gly-gly linker; SEQ ID NO: 19);

[0118] C34-3F1 l exp (the 3F11 nanobody linked to the C34 HIV fusion inhibitor peptide in the opposite orientation; SEQ ID NO: 20);Table 5Table 6Table 7Table 8

[0120] 3F1 l-C34-N10Q-exp (the 3F11 nanobody linked to the C34 HIV fusion inhibitor peptide through a gly-gly linker with a single point mutation (Asparagine to Glutamine (N-to-Q) introduced to avoid glycosylation; SEQ ID NO: 21);

[0121] 3F11-RIGID-C34_exp (the 3F11 nanobody linked to the C34 HIV fusion inhibitor peptide through a rigid peptide linker (EAAAKEAAAKEAAAK (SEQ ID NO: 17)); SEQ ID NO: 22); and,

[0122] 3F11-FLEX-C34_exp (the 3F11 nanobody linked to the C34 HIV fusion inhibitor peptide through a flexible peptide linker (GGGGSGGGGSGGGGS (SEQ ID NO: 18)); SEQ ID NO: 23).

[0123] These five recombinant constructs were purified and characterized by LCMS. Table 9 lists the expected and observed mass of the purified peptide constructs.

[0124] Table 9

[0125] For each of the five conjugates, HIV pseudovirus inhibition assays were run in triplicate. Table 10 lists the IC50 values (in nM) observed in each inhibition test and FIG. 9 shows a dose response for each of the five recombinant constructs as well as the C34 inhibitor peptide. These data demonstrate the efficacy of these recombinantly-expressed conjugates of this disclosure and additionally indicate an increase in efficacy for constructs prepared with a longer (either rigid or flexible) peptide linking sequence.

[0126] Table 10

[0127] The various features and processes described above may be used independently of one another, or may be combined in various ways. All possible combinations and subcombinations are intended to fall within the scope of this disclosure. In addition, certain method or process steps may be omitted in some implementations. The methods and processes described herein are also not limited to any particular sequence and the steps or states relating thereto can be performed in other sequences that are appropriate. For example, described steps or states may be performed in an order other than that specifically disclosed, or multiple steps or states may be combined in a single step or state. The example steps or states may be performed in serial, in parallel, or in some other manner. Steps or states may be added to or removed from the disclosed example embodiments. The example systems and components described herein may be configured differently than described. For example, elements may be added to, removed from, or rearranged compared to the disclosed example embodiments.

[0128] While certain example embodiments have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions disclosed herein. Thus, nothing in the foregoing description is intended to imply that any particular feature, characteristic, step, module, or state is necessary or indispensable. Indeed, the novel methods and materials described herein may be embodied in a variety of other forms; furthermore, various omissions, substitutions andchanges in the form of the methods and materials described herein may be made without departing from the spirit of the inventions disclosed herein. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of certain of the inventions disclosed herein.

Claims

CLAIMSWhat is claimed is:

1. A polypeptide construct comprising a nanobody (Nb) linked to a Human Immunodeficiency Virus (HIV), fusion-inhibitor (FI) peptide.

2. The polypeptide construct of claim 1, wherein the Nb binds to a T-cell surface protein.3 The polypeptide construct of claim 2, wherein the T-cell surface protein is a CD4 receptor4 The polypeptide construct of claim 2, wherein the T-cell surface protein is a CXCR4 coreceptor.5 The polypeptide construct of claim 2, wherein the T-cell surface protein is a major histocompatibility complex (MHC) protein.6 The polypeptide construct of claim 5, wherein the T-cell surface protein is an MHC class 1 (MHC-1) protein.7 The polypeptide construct of any one of claims 1-6, wherein the Nb is selected from the group consisting of Nb-3F11 (SEQ ID NO:4), Nb-CD4-bl (SEQ ID NO:8), Nb-281E10 (SEQ ID NO:5), Nb-281F12 (SEQ ID NO:6), and Nb-30 (SEQ ID NO:7).8 The polypeptide construct of claim 1, wherein the Nb binds to an HIV protein.9 The polypeptide construct of claim 8, wherein the HIV protein is an envelope (Env) protein.10 The polypeptide construct of claim 9, wherein the Nb binds to an Env protein domain selected from CD4 binding site (CD4bs) domain, V3 loop domain, gp41 heptad domain, and membrane proximate external region (MPER) domain.11 The polypeptide construct of claim 9, wherein the Env protein is gp41.12 The polypeptide construct of claim 9, wherein the Env protein is gpl20.13 The polypeptide construct of any one of claims 1-12, wherein the HIV Fusion Inhibitor (FI) peptide binds with an HIV protein.14 The polypeptide construct of claim 13, wherein the HIV protein is an envelope (Env) protein.15 The polypeptide construct of claim 14, wherein the Env protein is gp41 or gp!20.

16. The polypeptide construct of claim 15, wherein the FI binds a gp41 protein domain selected from the N-terminal heptad repeat of gp41, the fusion peptide domain of gp41, and the C- terminal heptad repeat of gp41.

17. The polypeptide construct of claim 13, wherein the FI is selected from C34 (SEQ ID NO:9) and N36 (SEQ ID NO: 12).

18. The polypeptide construct of any one of claims 1-17, wherein the Nb and the FI peptide are linked through a covalent linker.

19. The polypeptide construct of claim 18, wherein the covalent linker comprises at least one amino acid selected from Glycine, Serine, Alanine, Lysine, and Glutamate.

20. The polypeptide construct of claim 19, wherein the amino acid residues comprise at least two glycine residues.

21. The polypeptide construct of claim 18, wherein the linker comprises an amino acid sequence selected from the group consisting of LPETGGG (SEQ ID NO: 13); LPETGGGHHHHHH (SEQ ID NO: 14); GGSGGGS (SEQ ID NO: 15);GGSGGGSGGSGGGS (SEQ ID NO: 16); EAAAKEAAAKEAAAK (SEQ ID NO: 17); and, GGGGSGGGGSGGGGS (SEQ ID NO: 18).

22. The polypeptide construct of any one of claims 18-21, wherein the linker comprises one or more of an azide, a triazole, and an azide-derivatized dibenzo-azacyclooctyne (DBCO).

23. The polypeptide construct of claim 22, wherein the linker is an azide-derivatized dibenzo- azacyclooctyne (DBCO) linked to an LPETGG peptide.

24. The polypeptide construct of claim 22, wherein the linker comprises at least one polyethylene glycol (PEG).

25. The polypeptide construct of claim 22, wherein the linker is selected from the group consisting of:GGSGGGSGGSGGGS (SEQ ID NO: 16).

26. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide construct of any one of claims 1-25.

27. A method for treating a subject infected with HIV comprising administering to the subject at least one polypeptide construct of any one of claims 1-25, or a pharmaceutical composition of claim 26.

28. The method of claim 27, wherein the administration is effective to treat Acquired Immunodeficiency Syndrome (AIDS) in the subject.

29. The method of claim 27, wherein the administration is effective to inhibit or completely block viral entry of HIV into T-cells of the subject.

30. The method of claim 27, wherein the administration is effective to increase white blood cell count in the treated subject.

31. The method of claim 27, wherein the administration is effective to increase the number of CD4+ T-cells in the subject.

32. The method of claim 27, wherein the administration is effective to decrease the HIV-titer in the subject.

33. The method of claim 27, wherein the administration is effective to decrease opportunistic microbial infections in the subject.

34. The method of claim 27, wherein the administration causes only minimal undesirable side effects in the subject.

35. A method of preventing an HIV infection in an uninfected subject comprising administering to the uninfected subject at least one polypeptide construct of any one of claims 1-23, or a pharmaceutical composition of claim 24.

36. The method of claim 35, wherein the polypeptide construct prevents HIV infection in a subject who is HIV-negative.

37. The method of claim 36, wherein the administered polypeptide construct inhibits HIV fusion with CD4+ or CXCR4+ cells of the subject.

38. A method of inhibiting the onset or progression of an HIV infection or an HIV-associated disorder in a subject comprising administering to the uninfected subject at least one polypeptide construct of any one of claims 1-25, or a pharmaceutical composition of claim 26.

39. The method of claim 38, wherein the administered polypeptide construct inhibits fusion of HIV to CXCR4+ or CD4+ immune cells in the subject.

Images