Patent Grant
11149069
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 29, 2015, is named DFS-109_01_Sequence_Listing.txt and is 99,180 bytes in size.
Human immunodeficiency viruses (HIVs) are responsible for the pandemic of acquired immunodeficiency syndrome (AIDS) and related viruses cause similar conditions in other primates (e.g., collectively known as primate immunodeficiency viruses (PIVs). The envelope (Env) glycoprotein trimer (i.e., a trimer of gp120/gp41 protomers that forms the representative “spikes” on the surface of PIV particles) of a PIV is a membrane-fusing machine that mediates virus entry into host cells (Wyatt and Sodroski (1998) Science 280:1884-1888). Binding of the gp120 exterior envelope glycoprotein to CD4 and the chemokine receptor on target cells triggers conformational changes that allow the gp41 transmembrane envelope glycoprotein to fuse the viral and cell membranes. As the only virus-specific protein exposed on PIV particles or infected cell membrane, the envelope glycoprotein trimer represents the sole target for protective antibodies having neutralizing and/or other antiviral capabilities (Wyatt and Sodroski (1998) Science 280:1884-1888; Walker and Burton (2008) Science 320:760-764). Only about 10% of HIV-1-infected individuals generate broadly neutralizing antibodies (BNAbs), typically after 3-4 years of infection. Monoclonal antibodies that neutralize a broad range of HIV-1 variants have been derived from this subset of chronically infected humans. Some BNAbs passively protect monkeys from infection with viruses bearing HIV-1 Env, suggesting their potential utility in vaccine-elicited protection.
Attempts to elicit such protective antibodies having broad potency against divergent PIV species and/or strains have been unsuccessful to date, due to several factors. First, the structure of the envelope glycoproteins provides only a limited number of conserved sites accessible to antibodies. Second, antibodies must further target these limited epitopes with the correct angle-of-approach in order to bypass steric impediments (e.g., variable loops, glycans, and adjacent subunits) that interfere with antibody binding. Third, many candidate immunogens do not retain important neutralization epitopes in a stable manner. Even in cases where certain conformation-sensitive epitopes can be recognized by desired protective antibodies, these conformations are sampled in only a small percentage of the population of immunogen molecules at a given time due to the lability of the envelope glycoprotein trimer and dissociation of individual gp120 and gp41 subunits.
Indeed, a high percentage of the recently identified HIV-1-neutralizing monoclonal antibodies recognized Env epitopes in a quaternary structure- and/or N-linked carbohydrate-dependent manner that depends upon native envelope glycoprotein trimer structures. For example, the epitope for the broadly neutralizing human monoclonal antibody (mAb), b12, overlaps the CD4-binding site on gp120 and is present on monomeric gp120. However, b12 reacts far better with native, oligomeric gp120 than might be predicted from its monomer reactivity, which accounts for its unusually potent neutralization activity. Thus, the IgG1b12 epitope is oligomer-dependent, but not oligomer-specific. By contrast, many antibodies that are strongly reactive with CD4-binding site-related epitopes on monomeric gp120 fail to react with the native trimer and therefore do not neutralize primate immunodeficiency viruses.
Moreover, the rational design and modification of immunogens that overcome these hurdles has been hampered by a lack of structural information useful for generating soluble envelope glycoprotein trimers that faithfully mimic the native Env spike and function as effective immunogens for eliciting broadly protective antibodies. Although several crystal structures of monomeric HIV-1 gp120 core fragments in the CD4-bound state (Kwong et al. (1998) Nature 393:648-659; Huang et al. (2005) Science 310:1025-1028; Zhou et al. (2007) Nature 445:732-737; Huang et al. (2007) Science 317:1930-1934; Pancera et al. (2010) Proc. Natl. Acad. Sci. USA 107:1166-1171), trimeric gp41 ectodomain fragments in the post-fusion state (Weissenhorn et al. (1997) Nature 387:426-430; Chan et al. (1997) Cell 89:263-273; Buzon et al. (2010) PLoS Pathog. 6:e1000880), and unliganded monomeric gp120 core complexes from simian immunodeficiency virus (SIV) (Chen et al. (2005) Nature 433:834-841) have been determined at the atomic level, structural information is lacking on the unliganded state of gp120 and gp41 in the Env trimer and on the quaternary interactions that maintain the conformational integrity of the native trimer. Such information is critical for the rational design of conformationally stabilized quaternary PIV envelope glycoprotein trimers effective as immunogens for eliciting neutralizing responses (e.g., broadly neutralizing antibodies). Moreover, the currently defined gp120 and gp41 structures do not include a number of functionally important components (gp120 V1/V2 regions; gp41 fusion peptide, disulfide-bonded loop, transmembrane region and cytoplasmic tail; and glycans) that were artificially removed to facilitate crystallization. Although alternatives to X-ray crystallographic methods, such as cryo-electron microscopy (cryo-EM), have yielded electron density maps of purified Env variants at 18-30-Å resolution (Zhu et al. (2006) Nature 441:847-852; Zanetti et al. (2006) PLoS Pathog. 2:e83; Liu et al. (2008) Nature 455:109-113; White et al. (2010) PLoS Pathog. 6:e1001249; Wu et al. (2010) Proc. Natl. Acad. Sci. USA 107:18844-18849; Hu et al. (2011) J. Virol. 85:2741-2750), such models contradict each other and are insufficient for building an atomic model.
Accordingly, there is a great need to identify compositions and methods useful for generating stabilized PIV envelope glycoprotein trimers that can function effectively as immunogens for eliciting protective antibodies.
The present invention overcomes the longstanding difficulties in generating broadly neutralizing anti-primate immunodeficiency virus agents by providing stabilized envelope trimer complexes and polypeptides thereof that conformationally mimic the natural complexes and enhance immunogenicity.
In one aspect, a stabilized primate immunodeficiency virus (PIV) envelope polypeptide trimer complex is provided, wherein (a) each protomeric unit of the complex comprises a gp120 subunit and a gp41 subunit, or immunogenic fragments thereof, (b) one or more subunits of the complex comprises one or more mutated amino acid residues that increases the stability of the complex, and (c) the one or more mutated amino acid residues does not substantially alter the conformation of the native complex, in which the mutated amino acid residues are not altered. In one embodiment, the conformation of the native complex in which the mutated amino acid residues are not altered is the conformation shown in any one of
In another aspect, an isolated polypeptide is provided comprising the amino acid sequence of gp120 and gp41 subunits of a PIV envelope polypeptide, wherein (a) one or more subunits comprises one or more mutated amino acid residues that increases the stability of envelope polypeptide trimer complexes comprising the polypeptide and (b) the one or more mutated amino acid residues does not substantially alter the conformation of the native envelope polypeptide trimer complex, in which the mutated amino acid residues are not altered. In one embodiment, the native envelope polypeptide trimer complex in which the mutated amino acid residues are not altered is in the conformation shown in any one of
In still another aspect, an isolated nucleic acid which encodes polypeptides described herein is provided. In one embodiment, the nucleic acid comprises a nucleotide sequence set forth in Table 4, and further having one or more mutated residues selected from the group of residues listed in Tables 6-9 and combinations thereof; or a degenerate variant thereof. In another embodiment, the nucleic acid is operably linked to a promoter. In yet another aspect, a vector comprising a nucleic acid described herein is provided. In one embodiment, the vector is an expression vector. In another aspect, a host cell which comprises a vector described herein is provided. In one embodiment, the host cell is a mammalian cell having the ability to glycosylate proteins. In still another aspect, a method of producing a polypeptide comprising culturing a host cell in an appropriate culture medium to thereby produce the polypeptide is provided. In one embodiment, the host cell is a mammalian cell having the ability to glycosylate proteins.
In yet another aspect, an isolated broadly neutralizing antibody or antigen-binding portion thereof is provided that specifically binds to a trimeric complex or polypeptide described herein. In one embodiment, the antibody or antigen-binding portion thereof is a monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, single-chain antibody, antibody fragment, or is detectably labeled.
In another aspect, an immunogenic composition is provided comprising a trimeric complex or polypeptide described herein, and a pharmaceutically acceptable carrier. In one embodiment, the immunogenic composition further comprises an adjuvant. In another embodiment, the adjuvant is selected from the group consisting of alum, Freund's incomplete adjuvant, saponin, Quil A, QS-21, Ribi Detox, monophosphoryl lipid A, a CpG oligonucleotide, CRL-1005, L-121, and any combination thereof. In still another embodiment, the immunogenic composition is capable of eliciting primate immunodeficiency virus-specific neutralizing antibodies in mammals. In yet another embodiment, the PIV is SIV, HIV-1, or HIV-2.
In still another aspect, a method of generating stabilized PIV envelope polypeptide trimer complexes is provided, comprising (a) recombinantly modifying a nucleic acid encoding a PIV envelope polypeptide to encode one or more mutated residues selected from the group consisting of mutated residues listed in Tables 6-9 and combinations thereof, wherein the one or more mutated amino acid residues does not substantially alter the conformation of the native envelope polypeptide trimer complex, in which the mutated amino acid residues are not altered; (b) expressing the recombinant nucleic acid to produce PIV envelope polypeptides that form trimers. In one embodiment, the conformation of the native complex when the mutated amino acid residues are not mutated is the conformation shown in any one of
In yet another embodiment, a method of identifying broadly neutralizing anti-primate immunodeficiency virus antibodies is provided, comprising (a) administering an effective amount of an agent selected from the group consisting of a trimeric complex, polypeptide, nucleic acid, vector, host cell, and immunogenic composition described herein, to a subject to generate antibodies that neutralize primate immunodeficiency viruses heterologous to the virus strain or subtype from which the immunogen was derived and (b) isolating antibodies specific for the administered complex or composition.
In another embodiment, a method of identifying broadly neutralizing anti-primate immunodeficiency virus antibodies is provided, comprising (a) administering an effective amount of an agent selected from the group consisting of a trimeric complex, polypeptide, nucleic acid, vector, host cell, and immunogenic composition described herein, to B cells in an in vitro cell culture system to generate antibodies that neutralize the primate immunodeficiency viruses heterologous to the virus strain or subtype from which the immunogen was derived and (b) isolating antibodies specific for the administered complex or composition.
In still another embodiment, a method of making an isolated hybridoma which produces a broadly neutralizing antibody that specifically binds to a trimeric complex or polypeptide is provided, the method comprising: a) immunizing a mammal with an effective amount of an agent selected from the group consisting a trimeric complex, polypeptide, nucleic acid, vector, host cell, and immunogenic composition described herein; b) isolating splenocytes from the immunized mammal; c) fusing the isolated splenocytes with an immortalized cell line to form hybridomas; and d) screening individual hybridomas for production of an antibody which specifically binds with said trimeric complex or polypeptide thereof to isolate the hybridoma. In yet another aspect, an antibody produced by methods described herein are provided.
In another aspect, a method of eliciting an immune response in a subject against a primate immunodeficiency virus is provided, comprising administering to the subject a prophylactically or therapeutically effective amount of an agent selected from the group consisting of a trimeric complex, polypeptide, nucleic acid, vector, host cell, immunogenic composition, and antibody described herein, to thereby elicit the immune response. In one embodiment, the agent is administered in a single dose. In another embodiment, the agent is administered in multiple doses. In still another embodiment, the agent is administered as part of a heterologous prime-boost regimen. In yet another embodiment, the immune response comprises eliciting PIV-specific neutralizing antibodies in mammals. In another embodiment, the PIV is SIV, HIV-1, or HIV-2.
In still another aspect, a method of preventing a subject from becoming infected with a primate immunodeficiency virus is provided, comprising administering to the subject a prophylactically effective amount of an agent selected from the group consisting of a trimeric complex, polypeptide, nucleic acid, vector, host cell, immunogenic composition, and antibody described herein, thereby preventing the subject from becoming infected with the primate immunodeficiency virus. In one embodiment, the subject is exposed to the PIV. In another embodiment, administration of the agent elicits PIV-specific neutralizing antibodies in mammals. In still another embodiment, the PIV is SIV, HIV-1, or HIV-2.
In yet another aspect, a method for reducing the likelihood of a subject's becoming infected with a primate immunodeficiency virus is provided, comprising administering to the subject a prophylactically effective amount of an agent selected from the group consisting of a trimeric complex, polypeptide, nucleic acid, vector, host cell, immunogenic composition, and antibody described herein, thereby reducing the likelihood of the subject's becoming infected with the primate immunodeficiency virus. In one embodiment, the subject is exposed to the PIV. In another embodiment, administration of the agent elicits PIV-specific neutralizing antibodies in mammals. In still another embodiment, the PIV is SIV, HIV-1, or HIV-2.
In another aspect, a method for preventing or delaying the onset of, or slowing the rate of progression of, a primate immunodeficiency virus-related disease in a subject infected with a primate immunodeficiency virus is provided, comprising administering to the subject a therapeutically effective amount of an agent selected from the group consisting of a trimeric complex, polypeptide, nucleic acid, vector, host cell, immunogenic composition, and antibody described herein, thereby preventing or delaying the onset of, or slowing the rate of progression of, the primate immunodeficiency virus-related disease in the subject. In one embodiment, administration of the agent elicits PIV-specific neutralizing antibodies in mammals. In another embodiment, the PIV is SIV, HIV-1, or HIV-2.
Table 1 shows a list of interactions related to gp120-gp120 association and gp120 TAD stability.
Table 2 shows a list of interactions at the gp41-gp41 interface.
Table 3 shows a list of interactions at the gp120-gp41 interface.
Table 4 shows a list of representative Env nucleic acid and amino acid sequences from numerous primate immunodeficiency viruses.
Table 5 shows a list of interprotomer and intersubunit contacts within the HIV-1 Env trimer.
Table 6 shows a list of interprotomer bonds useful for increasing the stability of Env trimers.
Table 7 shows a list of intersubunit residue pairs useful for increasing the stability of Env trimers, especially using disulfide bonds.
Table 8 shows a list of intrasubunit residue pairs useful for increasing the stability of Env trimers, especially using disulfide bonds or promoting hydrophobic interactions.
Table 9 shows a list of residues useful for increasing the stability of Env trimers by increasing the solubility of the trimers.
The present invention is based in part on the elucidation of the structure of the fully glycosylated HIV-1 envelope trimer in its unliganded, pre-fusion state, including the complete ectodomain, the transmembrane region, and all of the peptide-proximal asparagine-linked glycans, by cryo-electron microscopy (cryo-EM). The structure reveals a dramatic conformational transition of gp120 between its unliganded and CD4-bound states, a torus-like fold of gp41 entirely different from its post-fusion conformation, and a conserved topology of the glycan shield. The structure of the trimer exhibits a spring-loaded mechanism that stores the free energy fueling virus entry. The structure further provides insights into virus-host interactions, mechanisms by which primate immunodeficiency viruses evade immune responses, and represents an atomic reference for inhibitor and vaccine design. Specifically, one or more amino acid residue changes, whose positions are numbered herein according to the envelope protein of the HIV-1 HXBc2 reference isolate and equivalent positions identified within envelope proteins from different PIV species, strains, or isolates, can be engineered to enhance the stability of PIV envelope glycoprotein trimers.
Some embodiments of the present invention are directed to envelope complexes in an immunogenic or antigenic conformation sufficient to elicit broadly neutralizing responses (e.g., production of broadly neutralizing antibodies). According to one aspect of the present invention, the sequences described and/or claimed herein can be altered or designed to maintain the same or a substantially similar amino acid sequence or protein in an immunogenic or antigenic conformation. In addition, the amino acid sequences or proteins of the present invention can be altered or modified according to methods known in the art to have different sequences yet still be capable of being placed in an immunogenic or antigenic conformation and/or having increased conformationally stability. It is to be understood that the specific amino acid sequences and proteins described herein include sequences and proteins that are substantially similar or homologous thereto or those that can be modified in a manner contemplated by those skilled in the art without departing from the spirit and operation of the present invention.
In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
The term “adjuvants” refers to any agent suitable for enhancing the immunogenicity of an antigen, such as protein and nucleic acid. Adjuvants suitable for use with protein-based immunogens are well known in the art and include, but are not limited to, alum, Freund's incomplete adjuvant (FIA), Saponin, Quil A, QS21, Ribi Detox, Monophosphoryl lipid A (MPL), and nonionic block copolymers such as L-121 (Pluronic; Syntex SAF). Methods of combining adjuvants with antigens are well known to those skilled in the art. Adjuvants can also be in particulate form. The antigen can be incorporated into biodegradable particles composed of poly-lactide-co-glycolide (PLG) or similar polymeric material. Such biodegradable particles are known to provide sustained release of the immunogen and thereby stimulate long-lasting immune responses to the immunogen. Other particulate adjuvants, include but are not limited to, micellular mixtures of Quil A and cholesterol known as immunostimulating complexes (ISCOMs) and aluminum or iron oxide beads. It is also known to those skilled in the art that cytotoxic T lymphocyte and other cellular immune responses are elicited when protein-based immunogens are formulated and administered with appropriate adjuvants, such as ISCOMs and micron-sized polymeric or metal oxide particles. Suitable adjuvants for nucleic acid-based vaccines include, but are not limited to, Quil A, interleukin-12 delivered in purified protein or nucleic acid form, short bacterial immunostimulatory nucleotide sequence, such as CpG-containing motifs, interleukin-2/Ig fusion proteins delivered in purified protein or nucleic acid form, oil in water micro-emulsions such as MF59, polymeric microparticles, cationic liposomes, monophosphoryl lipid A (MPL), immunomodulators such as Ubenimex, and genetically detoxified toxins such as E. coli heat labile toxin and cholera toxin from Vibrio. Such adjuvants and methods of combining adjuvants with antigens are well known to those skilled in the art. In addition, methods for combining antigens and particulate adjuvants are well known to those skilled in the art.
The term “amino acid” is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing. The names of the natural amino acids are abbreviated herein in accordance with the recommendations of IUPAC-IUB.
Unless otherwise specified herein, the terms “antibody” and “antibodies” broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives can comprise a protein or chemical moiety conjugated to an antibody. The term “antibody” as used herein also includes an “antigen-binding portion” of an antibody (or simply “antibody portion”). The term “antigen-binding portion,” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., envelope glycoprotein trimer complexes or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Still further, an antibody or antigen-binding portion thereof can be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein. Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies can also be fully human. The terms “monoclonal antibodies” and “monoclonal antibody composition”, as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.
The term “antigen-presenting cells” include, but are not limited to, dendritic cells, Langerhan cell, monocytes, macrophages, muscle cells and the like. In some embodiments, antigen presenting cells present an antigen, or an immunogenic part thereof, such as a peptide, or derivative and/or analogue thereof, in the context of major histocompatibility complex I or complex II, to other cells.
The term “binding” or “interacting” refers to an association, which can be a stable association, between two molecules, e.g., between a polypeptide of the invention and a binding partner, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions. Exemplary interactions include protein-protein, protein-nucleic acid, protein-small molecule, and small molecule-nucleic acid interactions.
The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
The term “body fluid” refers to fluids that are excreted or secreted from the body as well as fluid that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, and vomit). In some embodiments, media described herein can contain or comprise body fluids.
The term “broadly neutralizing” is well known in the art and refers to the ability of one or more agents (e.g., antibodies, aptamers, protein-binding nucleic acids, small molecules, etc.) to react with an infectious agent to destroy or greatly reduce the virulence of the infectious agent. The presence of such a response has the potential to prevent the establishment of infection and/or to significantly reduce the number of cells that become infected with a PIV, potentially delaying viral spread and allowing for a better control of viral replication in the infected host. A broadly neutralizing antibody against a PIV will typically bind a variety of different clades, groups or mutants of PIV. In some embodiments, the broadly neutralizing anti-PIV agent is an antibody that specifically binds to and neutralizes two, three, four, five, six, seven, eight, nine, ten or more clades and/or two or more groups of PIV (e.g., within HIV-1 or HIV-2).
The term “canonical glycosylation site” includes, but is not limited to, an Asn-X-Ser or Asn-X-Thr sequence of amino acids that defines a site for N-linkage of a carbohydrate. In addition, Ser or Thr residues not present in such sequences to which a carbohydrate can be linked through an O-linkage are canonical glycosylation sites. In the latter case of a canonical glycosylation site, a mutation of the Ser and Thr residue to an amino acid other than a serine or threonine will remove the site of O-linked glycosylation.
The term “CCR5” or “C—C chemokine receptor type 5” refers to a chemokine receptor which binds members of the C—C group of chemokines. At least two transcript variants encoding the same human CCR5 protein exist. The sequence of human CCR5 transcript variant 1, which encodes the longer of the two human CCR5 isoforms (i.e., isoform a), is available to the public at the GenBank database under NM_00579.3 and NP_000570.1. The sequence of human CCR5 transcript variant 2 differs in the 5′ untranslated region (UTR) compared to variant 1, while still encoding the same CCR5 protein and the sequences can be found under NM_001100168.1 and NP_001093638.1. Nucleic acid and polypeptide sequences of CCR5 orthologs in organisms other than humans are well known and include, for example, mouse CCR5 (NM_009917.5 and NP_034047.2), chimpanzee CCR5 (NM_001009046.1 and NP_001009046.1), rat CCR5 (NM_043960.3 and NP_446412.2), cow CCR5 (NM_001011672.2 and NP_001011672.2), dog CCR5 (NM_001012342.2 and NP_001012342.2), and chicken CCR5 (NM_001045834.1 and NP_001039299.1). As used herein, CCR5 includes extracellular portions of CCR5 capable of binding PIV envelope proteins.
The term “CD4” refers to a membrane glycoprotein of T lymphocytes that interacts with major histocompability complex class II antigens and is also a receptor for PIVs. The sequence of the human CD4 transcript variant 1 is available to the public at the GenBank database under NM_000616.4 and NP_000607.1. Nucleic acid and polypeptide sequences of CD4 orthologs in organisms other than humans are well known and include, for example, mouse CCR5 (NM_013488.2 and NP_038516.1), chimpanzee CD4 (NM_0010099043.1 and NP_001009043.1), rat CD4 (NM_012705.1 and NP_036837.1), cow CD4 (NM_001103225.1 and NP_001096695.1), dog CD4 (NM_001003252.1 and NP_001003252.1), and chicken CD4 (NM_204649.1 and NP_989980.1). As used herein, CD4 includes extracellular portions of CD4 capable of binding PIV envelope proteins. The extracellular domain of CD4 consists of four contiguous immunoglobulin-like regions (D1, D2, D3, and D4, see Sakihama et al., Proc. Natl. Acad. Sci. 92:6444, 1995; U.S. Pat. No. 6,117,655), and amino acids 1 to 183 have been shown to be involved in gp120 binding. For instance, a binding molecule or binding domain derived from CD4 would comprise a sufficient portion of the CD4 protein to mediate specific and functional interaction between the binding fragment and a native or viral binding site of CD4. One such binding fragment includes both the D1 and D2 extracellular domains of CD4 (D1D2 is also a fragment of soluble CD4 or sCD4 which is comprised of D1, D2, D3, and D4), although smaller fragments may also provide specific and functional CD4-like binding. The gp120-binding site has been mapped to D1 of CD4.
The term “chemoselective reaction” refers to reactions that may be used to stabilize peptides or polypeptides. For example, chemoselective ligation reactions that may be used to stabilize such compositions as described herein include, but are not limited to, reactions between amino acids of polypeptides described herein and involving: (i) an aldehyde/ketone and a hydrazide to form a hydrazone; (ii) a ketone and a aminoxy group to form an oxime; (iii) a ketone and a thiosemicarbazide to form a thiosemicarbazone; (iv) an aldehyde and a beta-amino thiol to form a thiazolidine; (v) a thiocarboxylate and a .alpha-halo carbonyl to form a thioester; (vi) a thioester and a N-terminal peptide cysteine to form an amide; (vii) a alkyl halide and a thiol to form a thioether; and, (viii) a maleimide and a thiol to form a thioether.
The term “complex” refers to an association between at least two moieties (e.g. chemical or biochemical) that have an affinity for one another. “Protein complex” or “polypeptide complex” refers to a complex comprising at least one or more polypeptides. In one embodiment, a complex comprises a trimer of protomeric units comprising a gp120 subunit and a gp41 subunit. Embodiments of complexes described herein can encompass other molecules (e.g., polypeptides) that can bind to the complex, such as an antibody.
The term “CXCR4” or “C—X—C chemokine receptor type 4” is a chemokine receptor which binds members of the C—X—C group of chemokines. At least two transcript variants encoding the same human CXCR4 protein exist. The sequence of human CXCR4 transcript variant 1, which encodes the longer of the two human CXCR4 isoforms (i.e., isoform a), is available to the public at the GenBank database under NM_001008540.1 and NP_001008540.1. The sequence of human CXCR4 transcript variant 2 differs in the 5′ untranslated region (UTR) and lacks an in-frame portion of the 5′ coding region compared to variant 1 and therefore encodes a smaller polypeptide having a shorter N-terminus relative to that of isoform 1. Such nucleic acid and protein sequences can be found under NM_003467.2 and NP_003458.1. Nucleic acid and polypeptide sequences of CXCR4 orthologs in organisms other than humans are well known and include, for example, mouse CXCR4 (NM_009911.3 and NP_034041.2), chimpanzee CXCR4 (NM_001009047.1 and NP_001009047.1), rat CXCR4 (NM_022205.3 and NP_071541.2), cow CXCR4 (NM_174301.3 and NP_776726.1), dog CXCR4 (NM_001048026.1 and NP_001041491.1), and chicken CXCR4 (NM_204617.2 and NP_989948.2). As used herein, CXCR4 includes extracellular portions of CXCR4 capable of binding the PIV envelope protein.
The term “effective amount” refers to an amount sufficient to achieve a desired result. For example, a “prophylactically effective amount” refers to an amount sufficient to reduce the likelihood of a disorder from occurring. In addition, a “therapeutically effective amount” refers to an amount effective to slow, stop or reverse the progression of a disorder.
The term “envelope,” “envelope glycoprotein,” or “Env” in reference to PIV polypeptides refers to certain polypeptides and/or complexes produced by such viruses. Generally, the env gene encodes gp160, which is proteolytically cleaved into gp120 and gp140. Gp120 binds to the CD4 receptor on a target cell that has such a receptor, such as, e.g., a T-helper cell. Gp41 is non-covalently bound to gp120, and provides the second step by which HIV enters the cell. It is originally buried within the viral envelope, but when gp120 binds to a CD4 receptor, gp120 changes its conformation causing gp41 to become exposed, where it can assist in fusion with the host cell. In some embodiments, the envelope glycoprotein includes a transmembrane gp41 or gp36 (e.g., “gp41/gp36”) subunit and an external, noncovalently associated gp120 subunit. The Env complex is expressed as a trimeric structure of three gp120/gp41 pairs (“protomers”) that mediates the multistep process of fusion of the virion envelope with the membrane of the target CD4+ T-cell. In some PIVs, the gp41 subunit is truncated to a gp36 subunit. Table 4 shows a list of representative Env nucleic acid and amino acid sequences from numerous primate immunodeficiency viruses.
As used herein, “exposure” to a PIV refers to contact with the PIV such that infection could result.
The term “gp41” or “gp41/gp36” denotes the retroviral transmembrane envelope glycoprotein 41 or glycoprotein 36 that is found in retroviruses, such as PIVs. With respect to PIVs, gp41 or gp36 is a highly immunogenic protein which elicits a strong and sustained antibody response in humans infected with HIV. The structure of gp41 from HIV-1 is described herein and is representative of other gp41 sequences described herein. The term includes, without limitation, (a) whole gp41 including the transmembrane and cytoplasmic domains; (b) gp41 ectodomain (gp41ECTO); (c) gp41 modified by deletion or insertion of one or more glycosylation sites; (d) gp41 modified so as to eliminate or mask the well-known immunodominant epitope; (e) a gp41 fusion protein; and (f) gp41 labeled with an affinity ligand or other detectable marker. As used herein, “ectodomain” means the extracellular region of a transmembrane protein exclusive of the transmembrane spanning and cytoplasmic regions.
The term “gp120” denotes the outer envelope protein found in retroviruses, such as PIVs. The envelope protein is initially synthesized as a longer precursor protein of 845-870 amino acids in size, designated gp160. Gp160 forms a homotrimer and undergoes glycosylation within the Golgi apparatus. It is then cleaved by a cellular protease into gp120 and gp41. Gp41 contains a transmembrane domain and remains in a trimeric configuration and it interacts with gp120 in a non-covalent manner. Gp120 contains most of the external, surface-exposed, domains of the envelope glycoprotein complex, and it is gp120 which binds both to the cellular CD4 receptor and to the cellular chemokine receptors (e.g., CCR5). Fragments similar to those defined for gp41 described herein are also contemplated.
The term “immunizing” refers to generating an immune response to an antigen in a subject. This can be accomplished, for example, by administering a primary dose of an immunogen to a subject, followed after a suitable period of time by one or more subsequent administrations of the immunogen, so as to generate in the subject an immune response against the immunogen. A suitable period of time between administrations of the immunogen can readily be determined by one skilled in the art, and is usually on the order of several weeks to months.
The term “immune response” is intended to include, but is not limited to, T and/or B cell responses, that is, cellular and/or humoral immune responses. The immune response of a subject can be determined by, for example, assaying antibody production, immune cell proliferation, the release of cytokines, the expression of cell surface markers, cytotoxicity, and the like. As used herein, the term “immune cell” is intended to include, but is not limited to, cells that are of hematopoietic origin and play a role in an immune response. Immune cells include, but are not limited to, lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
As used herein, the term “inhibit” includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. For example, a PIV-related infection or disease is “inhibited” if at least one symptom of the disease, such as viral load or low T cell count, is alleviated, terminated, slowed, or prevented. As used herein, PIV-related infection or disease is also “inhibited” if recurrence of a disease symptom is reduced, slowed, delayed, or prevented. Such an inhibition can affect a PIV-mediated activity (e.g., infection, fusion (e.g., target cell entry and/or syncytia formation), viral spread and the like) and/or a decrease in viral titer. For example, a PIV-mediated activity can be decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more. The term “promote,” in some embodiments, can be used in the exact opposite manner as “inhibit.”
The term “isolated polypeptide” refers to a polypeptide that (1) is not associated with proteins that it is normally found within nature, (2) is isolated from the cell in which it normally occurs, (3) is substantially free of other proteins from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature. The term “substantially free of cellular material” includes preparations of protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of envelope protein having less than about 30% (by dry weight) of non-envelope protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-envelope protein, still more preferably less than about 10% of non-envelope protein, and most preferably less than about 5% non-envelope protein. When the protein is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The language “substantially free of chemical precursors or other chemicals” includes preparations of envelope protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of envelope protein having less than about 30% (by dry weight) of chemical precursors of non-envelope chemicals, more preferably less than about 20% chemical precursors of non-envelope chemicals, still more preferably less than about 10% chemical precursors of non-envelope chemicals, and most preferably less than about 5% chemical precursors of non-envelope chemicals. In preferred embodiments, isolated proteins or biologically active portions thereof lack contaminating proteins from the same animal from which the envelope protein is derived. Typically, such proteins are produced by recombinant expression of, for example, a human envelope protein in a nonhuman cell. Similar considerations apply for “isolated nucleic acids.”
The term “not substantially altered,” “not substantially modulated,” and the like, unless otherwise defined, refers to a minimal deviation of a measured attribute in comparison to a reference control. The deviation can be measured according to quantitative or qualitative means. In one embodiment, the attribute's alteration is less than 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 1% or less different relative to the control (e.g., inter-residue differences, angles-of-approach, affinity for antibody binding, etc.).
The terms “label” or “labeled” refer to incorporation or attachment, optionally covalently or non-covalently, of a detectable marker into a molecule, such as a polypeptide. Various methods of labeling polypeptides are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes, fluorescent labels, heavy atoms, enzymatic labels or reporter genes, chemiluminescent groups, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). Examples and use of such labels are described in more detail below. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
An “overexpression” or “significantly higher level of expression or copy number” of a marker refers to an expression level or copy number in a test sample that is greater than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the marker in a control sample (e.g., sample from a healthy subject not afflicted with a PIV infection or related disease) and preferably, the average expression level or copy number of the marker in several control samples.
The term “primate immunodeficiency virus” or “PIV” refers to a group of well-known viruses infecting primates. The term includes human immunodeficiency viruses (HIV) and simian immunodeficiency viruses (SIV). For example, the term includes the human viruses, HIV-1 and HIV-2; the chimpanzee virus SIVcpz such as, for example, SIVcpzGab, SIVcpzCam, SIVcpzAnt, and SIVcpzUS; the sooty mangabey virus SIVsm; the African green monkey virus SIVagm such as, for example, SIVagm-1 and SIVagm-2; the mandrill virus SIVmnd such as, for example, SIVmnd14 and SIV mndGB 1, as well as a host of others including SIVsun/lhoest, SIVcol, SIVrcm, SIVsyk, SIVdeb, SIVgsn, SIVmon, SIVmus, and SIVtal. PIV is inclusive of all strains (e.g., SIVcpz) and sub-strains (e.g., SIVcpzGab). Regarding human immunodeficiency viruses, HIVs can be categorized into multiple clades with a high degree of genetic divergence. As used herein, the term “clade” refers to related human immunodeficiency viruses classified according to their degree of genetic similarity. There are currently three groups of HIV-1 isolates: M, N, and O. Group M (major strains) consists of at least ten clades, A through J. Group O (outer strains) can consist of a similar number of clades. Group N is a new HIV-1 isolate that has not been categorized in either group M or O.
The term “reducing the likelihood of a subject's becoming infected with a virus” refers to reducing the likelihood of the subject's becoming infected with the virus by at least two-fold. For example, if a subject has a 1% chance of becoming infected with the virus, a two-fold reduction in the likelihood of the subject becoming infected with the virus would result in the subject having a 0.5% chance of becoming infected with the virus. In one embodiment, reducing the likelihood of the subject's becoming infected with the virus means reducing the likelihood of the subject's becoming infected with the virus by at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold, or more.
The term “response to therapy” relates to any response of the PIV-related infection or disease to a therapy. Responses can be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD) or other qualitative criteria. Assessment of PIV-related infection or disease response can be done early after the onset of therapy, e.g., after a few hours, days, weeks or preferably after a few months. Additional criteria for evaluating the response to therapies are related to “survival,” which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality can be either irrespective of cause or tumor related); “recurrence-free survival” (e.g., viral load below a detectable threshold); metastasis free survival; disease free survival. The length of said survival can be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to therapy, probability of survival, probability of disease manifestation recurrence within a given time period. For example, in order to determine appropriate threshold values, a particular therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to viral load or other measurements that were determined prior to administration of any therapy. Alternatively, outcome measures, such as overall survival and disease-free survival can be monitored over a period of time for subjects following therapy for whom measurement values are known.
The term “stabilizing” or “enhancing stability” of an entity, such as a polypeptide or protein complex, means to make the entity more long-lived or resistant to dissociation. Enhancing stability can be achieved, for example, by enhancing covalent interactions, by enhancing non-covalent interactions, and/or reducing steric interactions. A covalent interaction is a chemical linkage between two atoms or radicals formed by the sharing of a pair of electrons (a single bond), two pairs of electrons (a double bond) or three pairs of electrons (a triple bond). Covalent interactions are also known in the art as electron pair interactions or electron pair bonds (e.g., disulfide bonds and chemoselective reactions). Non-covalent interactions include, but are not limited to ionic bonds (e.g., salt bridges), hydrogen bonds, hydrophobic interactions, van der Waals interactions, and weak chemical bonds (via short-range noncovalent interactions). A review of noncovalent interactions can be found in Alberts et al., in Molecular Biology of the Cell, 3rd Ed., Garland Publishing, 1994. Steric interactions are generally understood to include those where the structure of the compound is such that it is capable of occupying a site by virtue of its three dimensional structure, as opposed to any attractive forces between the compound and the site. Stabilizing interactions can include one or more of the interactions described herein, or any combination thereof. Stability-enhancing changes can be introduced by recombinant methods. As used herein, “mutant” means that which is not wild type, compared to a reference control.
The term “subject” refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a PIV-related infection or disease.
The term “substantially free of chemical precursors or other chemicals” includes preparations of antibody, polypeptide, peptide or fusion protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of antibody, polypeptide, peptide or fusion protein having less than about 30% (by dry weight) of chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, more preferably less than about 20% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, still more preferably less than about 10% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, and most preferably less than about 5% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals.
As used herein, the term “survival” includes all of the following: survival until mortality, also known as overall survival (wherein said mortality can be either irrespective of cause or tumor related); “recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); disease free survival (wherein the term disease shall include antiviral infection and diseases associated therewith). The length of said survival can be calculated by reference to a defined start point (e.g. time of diagnosis or start of treatment) and end point (e.g. death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of disease recurrence.
A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
A “transcribed polynucleotide” or “nucleotide transcript” is a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a marker of the invention and normal post-transcriptional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.
An “underexpression” or “significantly lower level of expression or copy number” of a marker refers to an expression level or copy number in a test sample that is greater than the standard error of the assay employed to assess expression or copy number, but is preferably at least twice, and more preferably three, four, five or ten or more times less than the expression level or copy number of the marker in a control sample (e.g., sample from a healthy subject not afflicted with PIV-related infection or disease) and preferably, the average expression level or copy number of the marker in several control samples.
The term “virally infected” refers to the introduction of viral genetic information into a target cell, such as by fusion of the target cell membrane with the virus or infected cell. The target can be a cell of a subject. In some embodiments, the target cell is a cell in a human subject.
There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.
An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet can be employed (illustrated above). Therefore, a number of different nucleotide sequences can code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms can translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine can be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.
In view of the foregoing, the nucleotide sequence of a DNA or RNA coding for a fusion protein or polypeptide of the invention (or any portion thereof) can be used to derive the fusion protein or polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for fusion protein or polypeptide amino acid sequence, corresponding nucleotide sequences that can encode the fusion protein or polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence). Thus, description and/or disclosure herein of a nucleotide sequence which encodes a fusion protein or polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence.
Similarly, description and/or disclosure of a fusion protein or polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
Before the present invention is further described, it will be appreciated that specific sequence identifiers (SEQ ID NOs) have been referenced throughout the specification for purposes of illustration and should therefore not be construed to be limiting. Any marker of the invention, including, but not limited to, the markers described in the specification and markers described herein are well known in the art and can be used in the embodiments of the invention.
It is further to be understood that this invention is not limited to particular embodiments described, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an envelope glycoprotein trimer complex” includes a plurality of such complexes and reference to “the active agent” includes reference to one or more active agents and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.
I. Isolated Nucleic Acids
One aspect of the invention pertains to isolated nucleic acid molecules that encode PIV envelope polypeptides having the ability to enhance the stability of envelop glycoprotein trimers in an immunologic conformation that enhances broadly neutralizing anti-PIV responses. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (i.e., cDNA or genomic DNA) and RNA molecules (i.e., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated envelope nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in viral DNA. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
A PIV envelope nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of an envelope polypeptide-encoding nucleic acid sequence shown in Table 4 that further encodes one or more mutated residues listed in Tables 6-9, or a nucleotide sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95%, 96%, 97%, 98%, 99% or more identical to such nucleotide sequences, can be engineered and isolated using standard molecular biology techniques and the sequence information provided herein (i.e., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, such nucleic acids can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon envelope sequences (e.g., the sequence of an envelope polypeptide-encoding nucleic acid sequence shown in Table 4 that further encodes one or more mutated residues listed in Tables 6-9, or fragment thereof, or the homologous nucleotide sequence). For example, RNA or DNA can be isolated from PIV nucleic acid (i.e., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNA can be prepared using reverse transcriptase (i.e., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for PCR amplification can be designed. A nucleic acid of the present invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to an envelope nucleotide sequence of the present invention can be prepared by standard synthetic techniques, i.e., using an automated DNA synthesizer.
Probes based on the envelope nucleotide sequences of the present invention can be used to detect homologs in related PIVs. In preferred embodiments, the probe further comprises a label group attached thereto, i.e., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which express an envelope protein, such as by measuring a level of an envelope-encoding nucleic acid in a sample of cells from a subject, i.e., detecting envelope RNA levels.
Nucleic acid molecules encoding other envelope members and thus which have a nucleotide sequence which differs from the envelope sequences of an envelope polypeptide-encoding nucleic acid sequence shown in Table 4 that further encodes one or more mutated residues listed in Tables 6-9, or fragment thereof, are contemplated. In one embodiment, the nucleic acid molecule(s) of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an envelope polypeptide amino acid sequence shown in Table 4 that further encodes one or more mutated residues listed in Tables 6-9, or fragment thereof, such that the protein or portion thereof forms a stable trimer complex that maintains or enhances one or more of the following biological activities: a) the conformation of the native trimer complex in the absence of the one or more mutated residues; b) the conformation of a trimer complex shown in any one of
In another embodiment, the nucleic acid encodes an envelope protein is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the entire amino acid sequence of an envelope polypeptide amino acid sequence shown in Table 4 that further encodes one or more mutated residues listed in Tables 6-9, or a fragment thereof.
The invention further encompasses envelope nucleic acid molecules that differ from the nucleotide sequences shown in an envelope polypeptide-encoding nucleic acid sequence shown in Table 4 that further encode one or more mutated residues listed in Tables 6-9. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an envelope polypeptide amino acid sequence shown in Table 4 that further has one or more mutated residues listed in Tables 6-9, or fragment thereof, or a protein having an amino acid sequence which is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to an envelope polypeptide amino acid sequence shown in Table 4 that further has one or more mutated residues listed in Tables 6-9, or fragment thereof.
It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of envelope can exist within a population (e.g., a mammalian and/or human population). Such genetic polymorphism in the envelope gene can exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding an envelope protein, preferably a mammalian, e.g., human, envelope protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the envelope gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in envelope that are the result of natural allelic variation and that do not alter the functional activity of envelope are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues within PV strains, clades, species, etc. can be isolated.
In addition to naturally-occurring allelic variants of the envelope sequence that can exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of an envelope polypeptide-encoding nucleic acid sequence shown in Table 4 that further encodes one or more mutated residues listed in Tables 6-9, or fragment thereof, thereby leading to changes in the amino acid sequence of the encoded envelope protein, without altering the functional ability of the envelope protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequences. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of an envelope polypeptide (e.g., an envelope polypeptide amino acid sequence shown in Table 4, or fragment thereof) without substantially altering the immunogenic conformation of envelope trimers, whereas an “essential” amino acid residue is affects the immunogenic conformation of the envelope trimer. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved between mouse and human) cannot be essential for activity and thus are likely to be amenable to alteration without altering envelope activity.
The term “sequence identity or homology” refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions compared×100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology. Unless otherwise specified “loop out regions”, e.g., those arising from, from deletions or insertions in one of the sequences are counted as mismatches.
The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. Preferably, the alignment can be performed using the Clustal Method. Multiple alignment parameters include GAP Penalty=10, Gap Length Penalty=10. For DNA alignments, the pairwise alignment parameters can be Htuple=2, Gap penalty=5, Window=4, and Diagonal saved=4. For protein alignments, the pairwise alignment parameters can be Ktuple=1, Gap penalty=3, Window=5, and Diagonals Saved=5.
In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available online), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available online), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0) (available online), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
An isolated nucleic acid molecule encoding an envelope protein homologous to an envelope polypeptide amino acid sequence shown in Table 4 that further has one or more mutated residues listed in Tables 6-9, or fragment thereof, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of an envelope polypeptide-encoding nucleic acid sequence shown in Table 4, or fragment thereof, or a homologous nucleotide sequence such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in envelope is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an envelope coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an envelope activity described herein to identify mutants that retain envelope activity. Following mutagenesis, the encoded protein can be expressed recombinantly (as described herein) and the activity of the protein can be determined using, for example, assays described herein.
Envelope protein levels can be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.
In some embodiments, PIV envelope expression levels are ascertained by measuring gene transcript (e.g., mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity. Expression levels can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.
In other embodiments, the envelope mRNA expression level can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term “biological sample” is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155).
The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays.
As an alternative to making determinations based on the absolute envelope expression level, determinations can be based on the normalized envelope expression level. Expression levels are normalized by correcting the absolute envelope expression level by comparing its expression to the expression of a non-envelope gene, e.g., a housekeeping or other reference gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a subject sample, to another sample, e.g., a normal sample, or between samples from different sources.
The level or activity of an envelope protein can also be detected and/or quantified by detecting or quantifying the expressed polypeptide. The envelope polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These can include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, and the like. A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express envelope.
II. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to the use of vectors, preferably expression vectors, containing a nucleic acid encoding envelope (or a portion or complex thereof). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. In one embodiment, adenoviral vectors comprising an envelope nucleic acid molecule are used.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
The recombinant expression vectors of the invention can be designed for expression of envelope in prokaryotic or eukaryotic cells. For example, envelope can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, and/or GST-thrombin cleavage site. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant envelope unfused to GST can be recovered by cleavage of the fusion protein with thrombin.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the envelope expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerivisae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
Alternatively, envelope can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny cannot, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, envelope protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Fao hepatoma cells, primary hepatocytes, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. In some embodiments, the host cell choice is determined according to the desire for glycosylation and, if so, the desired pattern of glycosylation. For example, trimers or polypeptides of the present invention can be produced using mammalian cell lines to produce polypeptides having mammalian patterns of glycosylation. Mammalian cell lines include, for example, monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line 293; baby hamster kidney cells (BHK); Chinese hamster ovary-cells-DHFR+ (CHO); Chinese hamster ovary-cells DHFR-(DXB11); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); mouse cell line (C127); and myeloma cell lines.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. An envelope polypeptide or fragment thereof, can be secreted and isolated from a mixture of cells and medium containing the polypeptide.
Alternatively, an envelope polypeptide or fragment thereof, can be retained cytoplasmically and the cells harvested, lysed and the protein or protein complex isolated. An envelope polypeptide or fragment thereof, can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of envelope or a fragment thereof. In other embodiments, heterologous tags can be used for purification purposes (e.g., epitope tags and FC fusion tags), according to standards methods known in the art.
Thus, a nucleotide sequence encoding all or a selected portion of an envelope polypeptide can be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the sequence into a polynucleotide construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, can be employed to prepare recombinant envelope polypeptides, or fragments thereof, by microbial means or tissue-culture technology in accord with the subject invention.
In another variation, protein production can be achieved using in vitro translation systems. In vitro translation systems are, generally, a translation system which is a cell-free extract containing at least the minimum elements necessary for translation of an RNA molecule into a protein. An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F). A variety of in vitro translation systems are well known in the art and include commercially available kits. Examples of in vitro translation systems include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, Ill.; and GIBCO/BRL, Grand Island, N.Y. In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes. In addition, an in vitro transcription system can be used. Such systems typically comprise at least an RNA polymerase holoenzyme, ribonucleotides and any necessary transcription initiation, elongation and termination factors. In vitro transcription and translation can be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs.
In certain embodiments, the envelope polypeptide, or fragment thereof, can be synthesized chemically, ribosomally in a cell free system, or ribosomally within a cell. Chemical synthesis can be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semi-synthesis through the conformationally-assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site. Full-length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products can be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules. (see e.g., U.S. Pat. Nos. 6,184,344 and 6,174,530; and T. W. Muir et al., Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science (1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245, p 616; L. H. Huang, et al., Biochemistry (1991): vol. 30, p 7402; M. Sclmolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193; K. Rajarathnam, et al., Science (1994): vol. 264, p 90; R. E. Offord, “Chemical Approaches to Protein Engineering”, in Protein Design and the Development of New therapeutics and Vaccines, J. B. Hook, G. Poste, Eds., (Plenum Press, New York, 1990) pp. 253-282; C. J. A. Wallace, et al., J. Biol. Chem. (1992): vol. 267, p 3852; L. Abrahmsen, et al., Biochemistry (1991): vol. 30, p 4151; T. K. Chang, et al., Proc. Natl. Acad. Sci. USA (1994) 91: 12544-12548; M. Schnlzer, et al., Science (1992): vol., 3256, p 221; and K. Akaji, et al., Chem. Pharm. Bull. (Tokyo) (1985) 33: 184).
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells can integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding envelope or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) envelope protein. Accordingly, the invention further provides methods for producing envelope protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding envelope has been introduced) in a suitable medium until envelope is produced. In another embodiment, the method further comprises isolating envelope from the medium or the host cell.
III. Isolated Envelope Polypeptides and Anti-Envelope Polypeptide/Trimer Antibodies
The present invention further provides isolated envelope polypeptides, or fragments thereof. In one aspect, an envelope polypeptide can comprise a full-length envelope amino acid sequence or a full-length envelope amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more 20 conservative amino acid substitutions. In one embodiment, the envelope polypeptides have an envelope polypeptide amino acid sequence shown in Table 4 that further has one or more mutated residues listed in Tables 6-9, or a fragment thereof. In another embodiment, the envelope polypeptides have an amino acid sequence that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identical to the entire amino acid sequence of an envelope polypeptide amino acid sequence shown in Table 4 that further has one or more mutated residues listed in Tables 6-9, or a fragment thereof. In addition, any envelope polypeptide of the present invention, or fragment thereof, forms a stable trimer complex that maintains or enhances one or more of the following biological activities: a) the conformation of the native trimer complex in the absence of the one or more mutated residues; b) the conformation of a trimer complex shown in any one of
In certain embodiments, an envelope polypeptide of the invention can be a fusion protein containing a domain which increases its solubility and bioavailability and/or facilitates its purification, identification, detection, and/or structural characterization. In such embodiments, the heterologous portions should not substantially alter the immunogenic conformation of envelope trimers. Exemplary domains, include, for example, glutathione S-transferase (GST), protein A, protein G, calmodulin-binding peptide, thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His, poly His-Asp or FLAG fusion proteins and tags. Additional exemplary domains include domains that alter protein localization in vivo, such as signal peptides, type III secretion system-targeting peptides, transcytosis domains, nuclear localization signals, etc. In various embodiments, an envelope polypeptide of the invention can comprise one or more heterologous fusions. Polypeptides can contain multiple copies of the same fusion domain or can contain fusions to two or more different domains. The fusions can occur in within the polypeptide as an in-frame insertion, at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or at both the N- and C-terminus of the polypeptide. It is also within the scope of the invention to include linker sequences between a polypeptide of the invention and the fusion domain in order to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein. In another embodiment, the polypeptide can be constructed so as to contain protease cleavage sites between the fusion polypeptide and polypeptide of the invention in order to remove the tag after protein expression or thereafter. Examples of suitable endoproteases, include, for example, Factor Xa and TEV proteases.
In some embodiments, envelope polypeptides, or fragments thereof, are fused to an antibody fragment (e.g., Fc polypeptides). Techniques for preparing these fusion proteins are known, and are described, for example, in WO 99/31241 and in Cosman et. al., 2001 Immunity 14:123 133. Fusion to an Fc polypeptide offers the additional advantage of facilitating purification by affinity chromatography over Protein A or Protein G columns.
In still another embodiment, an envelope polypeptide can be labeled with a fluorescent label to facilitate their detection, purification, or structural characterization. In an exemplary embodiment, an envelope polypeptide of the invention can be fused to a heterologous polypeptide sequence which produces a detectable fluorescent signal, including, for example, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Renilla reniformis green fluorescent protein, GFPmut2, GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), citrine and red fluorescent protein from discosoma (dsRED).
Fragments or biologically active portions of the envelope proteins can include polypeptides comprising amino acid sequences derived from the amino acid sequence of the envelope protein, e.g., an envelope polypeptide amino acid sequence shown in Table 4 that further has one or more mutated residues listed in Tables 6-9, or fragment thereof, which include fewer amino acids than the full-length envelope protein, and exhibit at least one activity of the envelope protein, or complex thereof. In one embodiment, an envelope protein can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more fewer amino acids, whether contiguous or not contiguous. For example, deletion or replacement of certain sequences (e.g., the proteolytic cleavage site, signal sequence, and the like) that do not substantially affect the immunogenic conformation of native envelope trimers are contemplated.
Envelope proteins described herein can be produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the envelope protein is expressed in the host cell. The envelope protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, an envelope protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native envelope protein can be isolated from cells (e.g., lymphoma cells), for example, using an anti-envelope antibody (described further below).
The present invention further provides envelope trimer complexes formed by the envelope polypeptides of the present invention. In one embodiment, either envelope proteins or trimer complexes thereof (e.g., envelope glycoprotein trimer complexes), can be used as immunogens to generate neutralizing agents (e.g., antibodies, aptamers, and the like) that bind envelope polypeptides or trimer complexes thereof, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length envelope protein can be used or, alternatively, antigenic peptide fragments of envelope, or peptides in complex, can be used as immunogens. An envelope polypeptide or trimer thereof of the present invention can be used to prepare antibodies by immunizing a suitable subject, (e.g., human, monkey, rabbit, goat, mouse or other mammal) with the immunogen as further described herein. An appropriate immunogenic preparation can contain, for example, recombinantly expressed envelope protein or a chemically synthesized envelope polypeptide or trimer thereof. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic envelope polypeptide or trimer thereof that induces a polyclonal anti-envelope antibody response.
Accordingly, another aspect of the invention pertains to the use of anti-envelope antibodies. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as envelope. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind envelope. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of envelope. A monoclonal antibody composition thus typically displays a single binding affinity for a particular envelope protein with which it immunoreacts.
Polyclonal anti-envelope antibodies can be prepared as described above by immunizing a suitable subject with an envelope immunogen, or fragment thereof. The anti-envelope antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized envelope. If desired, the antibody molecules directed against envelope can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, i.e., when the anti-envelope antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an envelope immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds envelope.
Any of the many well-known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-envelope monoclonal antibody (see, i.e., G. Galfre et al. (1977) Nature 266:550-52; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, i.e., the P3-NS1/1Ag4-1, P3-x63Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind envelope, i.e., using a standard ELISA assay.
Additionally, recombinant anti-envelope polypeptide and/or anti-envelope trimer antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.
An anti-envelope polypeptide and/or anti-envelope trimer antibody (e.g., monoclonal antibody) can be used to isolate and/or detect (e.g., in diagnostic assays) envelope polypeptides by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-envelope polypeptide and/or anti-envelope trimer antibody can facilitate the purification of natural envelope polypeptides from cells and of recombinantly produced envelope expressed in host cells. Moreover, an anti-envelope polypeptide and/or anti-envelope trimer antibody can be used to detect envelope proteins or trimers thereof (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the envelope protein. In some embodiments, for example, such antibodies can be used in quantitative immunohistochemical assays to determine PIV viral loads. Thus, anti-envelope antibodies can be used to monitor protein levels in a cell or tissue, e.g., cells or tissues infected with a PIV, as part of a clinical testing procedure, e.g., in order to monitor the efficacy of an anti-PIV therapy. Detection can be facilitated by coupling (e.g., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.
In vivo techniques for detection of envelope protein include introducing into a subject a labeled antibody directed against the protein. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
IV. Identification of Agents that Modulate Envelope Polypeptides and Trimers Thereof
The envelope nucleic acid and polypeptide molecules described herein can be used to design modulators of one or more of biological activities of the complex or complex polypeptides. In particular, information useful for the design of therapeutic and diagnostic molecules, including, for example, the protein domain, structural information, and the like for polypeptides of the present invention is described herein.
In one aspect, modulators, inhibitors, or antagonists directed against the envelope polypeptides of the present invention and trimers thereof and biological complexes containing them (e.g., natural or synthetic lipid membranes containing envelope trimers) can be used for therapeutic, prognostic, and diagnostic purposes. In certain exemplary embodiments, screening assays for identifying modulators, i.e., candidate or test compounds or agents (e.g., antibodies, peptides, cyclic peptides, peptidomimetics, small molecules, small organic molecules, or other drugs) which have an inhibitory effect on envelope polypeptides or trimers thereof and/or one or more PIV-mediated activities described herein are provided.
Modulators of envelope polypeptides and trimers thereof can be identified and developed as described herein using techniques and methods known to those of skill in the art. The modulators of the invention can be employed, for instance, to inhibit and treat PIV infections or PIV-mediated disorders. The modulators of the invention can elicit a change in one or more of the following activities: (a) a change in the level and/or rate of formation of an envelope trimer complex, (b) a change in the activity of an envelope nucleic acid and/or polypeptide, (c) a change in the stability of an envelope nucleic acid or polypeptide/trimer, (d) a change in the conformation of an envelope nucleic acid or polypeptide/trimer, or (e) a change in the activity of at least one polypeptide contained in an envelope trimer complex. A number of methods for identifying a molecule which modulates an envelope nucleic acid and/or polypeptide are known in the art. For example, in one such method, an envelope nucleic acid or polypeptide/trimer is contacted with a test compound and the activity of the envelope nucleic acid or polypeptide/trimer is determined in the presence of the test compound, wherein a change in the activity of the envelope nucleic acid and/or polypeptide in the presence of the compound as compared to the activity in the absence of the compound (or in the presence of a control compound) indicates that the test compound modulates the activity of the envelope nucleic acid and/or polypeptide.
Compounds to be tested for their ability to act as modulators of envelope nucleic acids or polypeptide/trimer, can be produced, for example, by bacteria, yeast or other organisms (e.g. natural products), produced chemically (e.g. small molecules, including peptidomimetics), or produced recombinantly. Compounds for use with the above-described methods can be selected from the group of compounds consisting of lipids, carbohydrates, polypeptides, peptidomimetics, peptide-nucleic acids (PNAs), small molecules, natural products, aptamers and polynucleotides. In certain embodiments, the compound is a polynucleotide. In certain embodiments, the compound comprises a biologically active fragment of an envelope polypeptide (e.g., a dominant negative form that binds to, but does not activate, envelope).
A variety of assay formats will suffice and, in light of the present disclosure, those not expressly described herein can nevertheless be comprehended by one of ordinary skill in the art based on the teachings herein. Assay formats for analyzing envelope trimer complex formation and/or activity of an envelope nucleic acid and/or polypeptide can be generated in many different forms and include assays based on cell-free systems, e.g. purified proteins or cell lysates, as well as cell-based assays which utilize intact cells. Simple binding assays can also be used to detect agents which modulate an envelope nucleic acid or polypeptide/trimer, for example, by enhancing the formation of an envelope trimer complex and/or by enhancing the binding of an envelope polypeptide to trimer complex to a substrate. Another example of an assay useful for identifying a modulator of an envelope is a competitive assay that combines one or more envelope polypeptides with a potential modulator, such as, for example, polypeptides, nucleic acids, natural substrates or ligands, antibodies, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. Envelope polypeptides/trimers can be labeled, such as by radioactivity or a colorimetric compound, such that envelope complex formation and/or activity can be determined accurately to assess the effectiveness of the potential modulator.
Assays can employ kinetic or thermodynamic methodology using a wide variety of techniques including, but not limited to, microcalorimetry, circular dichroism, capillary zone electrophoresis, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and combinations thereof. Assays can also employ any of the methods for isolating, preparing and detecting envelopes polypeptides or complexes, as described above.
Complex formation between an envelope polypeptide, or fragment thereof, and a binding partner (e.g., CD4, CCR5, or CXCR) can be detected by a variety of methods. Modulation of the complex's formation can be quantified using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled polypeptides or binding partners, by immunoassay, or by chromatographic detection. Methods of isolating and identifying envelop trimer complexes described above can be incorporated into the detection methods.
In certain embodiments, it can be desirable to immobilize an envelope polypeptide to facilitate separation of envelope trimer complexes from uncomplexed enveloped polypeptides, as well as to accommodate automation of the assay. In any case, binding of an envelope polypeptide or a trimer complex thereof to a binding partner can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/polypeptide (GST/polypeptide) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the binding partner, e.g. an 35S-labeled binding partner, and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions can be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of envelope polypeptides found in the bead fraction quantified from the gel using standard electrophoretic techniques such as described in the appended examples.
Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, an envelope polypeptide or trimer thereof can be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated polypeptide molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptide can be derivatized to the wells of the plate, and polypeptide trapped in the wells by antibody conjugation. As above, preparations of a binding partner and a test compound are incubated in the polypeptide presenting wells of the plate, and the amount of complex trapped in the well can be quantified. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the binding partner, or which are reactive with the envelope polypeptide and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity. In the instance of the latter, the enzyme can be chemically conjugated or provided as a fusion protein with the binding partner.
Antibodies against the envelope polypeptide can be used for immunodetection purposes. Alternatively, the envelope polypeptide to be detected can be “epitope-tagged” in the form of a fusion protein that includes, in addition to the polypeptide sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources). For instance, the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharmacia, N.J.).
In certain in vitro embodiments of the present assay, the protein or the set of proteins engaged in a protein-protein, protein-substrate, or protein-nucleic acid interaction comprises a reconstituted protein mixture of at least semi-purified proteins. By semi-purified, it is meant that the proteins utilized in the reconstituted mixture have been previously separated from other cellular or viral proteins. For instance, in contrast to cell lysates, the proteins involved in a protein-substrate, protein-protein or nucleic acid-protein interaction are present in the mixture to at least 50% purity relative to all other proteins in the mixture, and more preferably are present at 90-95% purity. In certain embodiments of the subject method, the reconstituted protein mixture is derived by mixing highly purified proteins such that the reconstituted mixture substantially lacks other proteins (such as of cellular or viral origin) which might interfere with or otherwise alter the ability to measure activity resulting from the given protein-substrate, protein-protein interaction, or nucleic acid-protein interaction.
In one embodiment, the use of reconstituted protein mixtures allows more careful control of the protein-substrate, protein-protein, or nucleic acid-protein interaction conditions. Moreover, the system can be derived to favor discovery of modulators of particular intermediate states of the protein-protein interaction. For instance, a reconstituted protein assay can be carried out both in the presence and absence of a candidate agent, thereby allowing detection of a modulator of a given protein-substrate, protein-protein, or nucleic acid-protein interaction.
Assaying biological activity resulting from a given protein-substrate, protein-protein or nucleic acid-protein interaction, in the presence and absence of a candidate modulator, can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes.
In still further embodiments, the envelope polypeptide or trimer complex thereof can be generated in whole cells, taking advantage of cell culture techniques to support the subject assay. For example, the envelope polypeptide or trimer complex thereof, can be constituted in a prokaryotic or eukaryotic cell culture system. This allows for an environment more closely approximating that which therapeutic use of the modulator would require, including the ability of the agent to gain entry into the cell. Furthermore, certain of the in vivo embodiments of the assay are amenable to high through-put analysis of candidate agents.
Some or all of the components can be derived from exogenous sources. For instance, fusion proteins can be introduced into the cell by recombinant techniques (such as through the use of an expression vector), as well as by microinjecting the fusion protein itself or mRNA encoding the fusion protein. Moreover, in the whole cell embodiments of the subject assay, the reporter gene construct can provide, upon expression, a selectable marker. Such embodiments of the subject assay are particularly amenable to high through-put analysis in that proliferation of the cell can provide a simple measure of the protein-protein interaction.
The amount of transcription from the reporter gene can be measured using any method known to those of skill in the art to be suitable. For example, specific mRNA expression can be detected using Northern blots or specific protein product can be identified by a characteristic stain, western blots or an intrinsic activity. In certain embodiments, the product of the reporter gene is detected by an intrinsic activity associated with that product. For instance, the reporter gene can encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence.
In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays of the present invention which are performed in cell-free systems, such as can be derived with purified or semi-purified proteins or with lysates, are often preferred as “primary” screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as can be manifest in an alteration of binding affinity with other proteins or changes in enzymatic properties of the molecular target. Accordingly, potential modulators of envelope can be detected in a cell-free assay generated by constitution of a functional envelope polypeptide or trimer complex thereof in a cell lysate. In an alternate format, the assay can be derived as a reconstituted protein mixture which, as described below, offers a number of benefits over lysate-based assays.
The activity of an envelope polypeptide or trimer complex thereof can be identified and/or assayed using a variety of methods well known to the skilled artisan. For example, the activity of an envelope nucleic acid and/or polypeptide can be determined by assaying for the level of expression of RNA and/or protein molecules. Transcription levels can be determined, for example, using Northern blots, hybridization to an oligonucleotide array or by assaying for the level of a resulting protein product. Translation levels can be determined, for example, using Western blotting or by identifying a detectable signal produced by a protein product (e.g., fluorescence, luminescence, enzymatic activity, etc.). Depending on the particular situation, it can be desirable to detect the level of transcription and/or translation of a single gene or of multiple genes. Similarly, it can be desirable to determine the status of stability-enhancing modifications made to the envelope polypeptides of the present invention (e.g., analyzing the production of disulfide bonds in Cys-Cys modifications and the like).
In other embodiments, the biological activity of an envelope nucleic acid and/or polypeptide can be assessed by monitoring changes in the phenotype of a targeted cell. For example, the detection means can include a reporter gene construct which includes a transcriptional regulatory element that is dependent in some form on the level and/or activity of an envelope nucleic acid and/or polypeptide. The envelope nucleic acid and/or polypeptide can be provided as a fusion protein with a domain that binds to a DNA element of a reporter gene construct. The added domain of the fusion protein can be one which, through its DNA binding ability, increases or decreases transcription of the reporter gene. Whichever the case can be, its presence in the fusion protein renders it responsive to an envelope nucleic acid and/or polypeptide. Accordingly, the level of expression of the reporter gene will vary with the level of expression of an envelope nucleic acid and/or polypeptide.
Moreover, in the whole cell embodiments of the subject assay, the reporter gene construct can provide, upon expression, a selectable marker. A reporter gene includes any gene that expresses a detectable gene product, which can be RNA or protein. Preferred reporter genes are those that are readily detectable. The reporter gene can also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties. For instance, the product of the reporter gene can be an enzyme which confers resistance to an antibiotic or other drug, or an enzyme which complements a deficiency in the host cell (i.e. thymidine kinase or dihydrofolate reductase). To illustrate, the aminoglycoside phosphotransferase encoded by the bacterial transposon gene Tn5 neo can be placed under transcriptional control of a promoter element responsive to the level of an envelope nucleic acid and/or polypeptide present in the cell. Such embodiments of the subject assay are particularly amenable to high through-put analysis in that proliferation of the cell can provide a simple measure of inhibition of the envelope nucleic acid and/or polypeptide.
V. Methods of the Invention
a. Methods of using immunogens
In one aspect, the present invention provides compositions useful as immunogens (e.g., trimeric complexes, polypeptides, nucleic acids, vectors, host cells, immunogenic compositions, and the like described herein). Such immunogens can be administered in any of a number of routes known in the art (e.g., to be compatible with eliciting strong antibody responses). Administration can be by injection, infusion, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes. Preferably, the composition is administered by intravenous, subcutaneous, intradermal, or intramuscular routes, or any combination thereof. The immunization protocol can include at least one priming dose, followed by one or multiple boosting doses administered over time. An exemplary range for an immunogenically effective amount of the present immunogenic polypeptides is about 5 to about 500 μg/kg body weight. A preferred range is about 10-100 μg/kg.
In one embodiment, one or more unit doses of the immunogen are given at one, two or three time points. The optimal number and timing of boosts can readily be determined using routine experimentation. Exemplary “prime-boost” regimens are described in U.S. Pat. No. 6,210,663 and WO 00/44410. In one embodiment, a priming composition is a replication-competent or replication-defective recombinant virus containing a nucleic acid molecule encoding the antigen, or a viral-like particle. In one particular embodiment, the priming composition is a non-replicating recombinant virus or viral-like particle derived from a PIV.
One method according to the present invention involves “priming” a mammalian subject by administration of a priming composition. “Priming,” as used herein, means any method whereby a first immunization using an antigen permits the generation of an immune response to the antigen upon a second immunization with the same antigen, wherein the second immune response is greater than that achieved where the first immunization is not provided.
Preferably, a boosting composition is administered about 2 to 27 weeks after administering the priming composition to a mammalian subject. The administration of the boosting composition is accomplished using an effective amount of a boosting composition containing or capable of delivering the same antigen as administered by the priming composition. As used herein, the term “boosting composition” includes, as one embodiment, a composition containing the same antigen as in the priming composition or precursor thereof, but in a different form, in which the boosting composition induces an immune response in the host. In one particular embodiment, the boosting composition comprises a recombinant soluble protein.
In one embodiment, a boosting composition is a replication-competent or replication-defective recombinant virus containing the DNA sequence encoding the protein antigen. In another embodiment, the boosting composition is a non-replicating alphavirus comprising a nucleic acid molecule encoding the protein antigen or a non-replicating vaccine replicon particle derived from an alphavirus. Adenoviruses, which naturally invade their host through the airways, infect cells of the airways readily upon intranasal application and induce a strong immune response without the need for adjuvants. In another embodiment, the boosting composition comprises a replication-defective recombinant adenovirus. Another example of a boosting composition is a bacterial recombinant vector containing the DNA sequence encoding the antigen in operable association with regulatory sequences directing expression of the antigen in tissues of the mammal. One example is a recombinant BCG vector. Other examples include recombinant bacterial vectors based on Salmonella, Shigella, and Listeria, among others. Still another example of a boosting composition is a naked DNA sequence encoding the antigen in operable association with regulatory sequences directing expression of the antigen in tissues of the mammal but containing no additional vector sequences. These vaccines can further contain pharmaceutically suitable or physiologically acceptable carriers. In still additional embodiments, the boosting compositions can include proteins or peptides (intact and denatured), heat-killed recombinant vaccines, inactivated whole microorganisms, antigen-presenting cells pulsed with the instant proteins or infected/transfected with a nucleic acid molecule encoding same, and the like, all with or without adjuvants, chemokines and/or cytokines.
Representative forms of antigenic immunogens include a “naked” DNA plasmid, a “naked” RNA molecule, a DNA molecule packaged into a replicating or nonreplicating viral vector, an RNA molecule packaged into a replicating or nonreplicating viral vector, a DNA molecule packaged into a bacterial vector, or proteinaceous forms of the antigen alone or present in virus-like particles, or combinations thereof.
In one embodiment, recombinant envelope polypeptides and trimers thereof, can be administered to subjects. In some embodiments, fusion proteins can be constructed and administered which have enhanced biological properties. In addition, the polypeptides can be modified according to well-known pharmacological methods in the art (e.g., pegylation, glycosylation, oligomerization, etc.) in order to further enhance desirable biological activities, such as increased immunogenicity, bioavailability, and/or decreased proteolytic degradation.
In one embodiment, “virus-like particles” or “VLPs” can be used, which are non-infectious particles in any host and do not contain all of the protein components of live virus particles. In one embodiment, VLPs contain the stabilized envelope glycoprotein trimers or polypeptides described herein and form membrane-enveloped virus-like particles. The advantages of using VLPs include (1) their particulate and multivalent nature, which is immunostimulatory, and (2) their ability to present the disulfide-stabilized envelope glycoproteins in a near-native, membrane-associated form. VLPs are produced by co-expressing the viral proteins (e.g., stabilized envelope glycoprotein trimers or polypeptides described herein) in the same cell. This can be achieved by any of several means of heterologous gene expression that are well-known to those skilled in the art, such as transfection of appropriate expression vector(s) encoding the viral proteins, infection of cells with one or more recombinant viruses (e.g., vaccinia) that encode the VLP proteins, or retroviral transduction of the cells. A combination of such approaches can also be used. The VLPs can be produced either in vitro or in vivo. VLPs can be produced in purified form by methods that are well-known to the skilled artisan, including centrifugation, as on sucrose or other layering substance, and by chromatography.
For embodiments using instant nucleic acid delivery, any means for the introduction of a polynucleotide into a subject, such as a human or non-human mammal, or cells thereof can be adapted to the practice of this invention for the delivery of the various constructs of the invention into the intended recipient. In one embodiment of the invention, the DNA constructs are delivered to cells by transfection, i.e., by delivery of “naked” DNA or in a complex with a colloidal dispersion system. A colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a lipid-complexed or liposome-formulated DNA. In the former approach, prior to formulation of DNA, e.g., with lipid, a plasmid containing a transgene bearing the desired DNA constructs can first be experimentally optimized for expression (e.g., inclusion of an intron in the 5′ untranslated region and elimination of unnecessary sequences (Felgner, et al., Ann NY Acad Sci 126-139, 1995). Formulation of DNA, e.g. with various lipid or liposome materials, can then be effected using known methods and materials and delivered to the recipient mammal. See, e.g., Canonico et al., Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al., Am J Physiol 268; Alton et al., Nat Genet. 5:135-142, 1993; and U.S. Pat. No. 5,679,647 by Carson et al.
The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs, which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
The surface of the targeted delivery system can be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand. Naked DNA or DNA associated with a delivery vehicle, e.g., liposomes, can be administered to several sites in a subject (see below).
Nucleic acids can be delivered in any desired vector. These include viral or non-viral vectors, including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). Nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels, including in virus particles, in liposomes, in nanoparticles, and complexed to polymers.
The nucleic acids encoding a protein or nucleic acid of interest can be in a plasmid or viral vector, or other vector as is known in the art. Such vectors are well known and any can be selected for a particular application. In one embodiment of the invention, the gene delivery vehicle comprises a promoter and a demethylase coding sequence. Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidylate synthase promoters. Other preferred promoters include promoters which are activatable by infection with a virus, such as the α- and β-interferon promoters, and promoters which are activatable by a hormone, such as estrogen. Other promoters which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter. A promoter can be constitutive or inducible.
In another embodiment, naked polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/11092 and U.S. Pat. No. 5,580,859. Such gene delivery vehicles can be either growth factor DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curiel et al., Hum. Gene. Ther. 3:147-154, 1992. Other vehicles which can optionally be used include DNA-ligand (Wu et al., J. Biol. Chem. 264:16985-16987, 1989), lipid-DNA combinations (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413 7417, 1989), liposomes (Wang et al., Proc. Natl. Acad. Sci. 84:7851-7855, 1987) and microprojectiles (Williams et al., Proc. Natl. Acad. Sci. 88:2726-2730, 1991).
A gene delivery vehicle can optionally comprise viral sequences, such as a viral origin of replication or packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus. In a preferred embodiment, the growth factor gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al., Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci. USA 81:6349, 1984, Miller et al., Human Gene Therapy 1:5-14, 1990, U.S. Pat. Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos. WO 89/02,468, WO 89/05,349, and WO 90/02,806. Numerous retroviral gene delivery vehicles can be utilized in the present invention, including for example those described in EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993; Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J. Neurosurg. 79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP 0,345,242 and WO91/02805).
Other viral vector systems that can be used to deliver a polynucleotide of the invention have been derived from herpes virus, e.g., Herpes Simplex Virus (U.S. Pat. No. 5,631,236 by Woo et al., issued Can 20, 1997 and WO 00/08191 by Neurovex), vaccinia virus (Ridgeway (1988) Ridgeway, “Mammalian expression vectors,” In: Rodriguez R L, Denhardt D T, ed. Vectors: A survey of molecular cloning vectors and their uses. Stoneham: Butterworth; Baichwal and Sugden (1986) “Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes,” In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press; Coupar et al. (1988) Gene, 68:1-10), and several RNA viruses. Preferred viruses include an alphavirus, a poxvirus, an arena virus, a vaccinia virus, a polio virus, and the like. They offer several attractive features for various mammalian cells (Friedmann (1989) Science, 244:1275-1281; Ridgeway, 1988, supra; Baichwal and Sugden, 1986, supra; Coupar et al., 1988; Horwich et al. (1990) J. Virol., 64:642-650).
Immunogens can be administered together with an adjuvant or other immunostimulant. Thus, the immunogens can further comprise one or more adjuvants or immunostimulating agents, which are preferably added to the fusion protein immunogens using for boosting the immune response. An adjuvant is any substance that can be added to an immunogen or to a vaccine formulation to enhance the immune-stimulating properties of the immunogenic moiety, such as a protein or polypeptide. Liposomes are also considered to be adjuvants. See, for example, Gregoriades, G. et al., Immunological Adjuvants and Vaccines, Plenum Press, New York, 1989; Michalek, S. M. et al., Liposomes as Oral Adjuvants, Curr. Top. Microbiol. Immunol. 146:51-58 (1989). Examples of adjuvants or agents that can add to the effectiveness of immunogens include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, and oil-in-water emulsions. Other adjuvants are muramyl dipeptide (MDP) and various MDP derivatives and formulations, e.g., N-acetyl-D-glucosaminyl-(β,1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) (Hornung, R L et al., Ther Immunol 1995 2:7-14) or ISAF-1 (5% squalene, 2.5% pluronic L121, 0.2% Tween 80 in phosphate-buffered solution with 0.4 mg of threonyl-muramyl dipeptide; see Kwak, L W et al., (1992) N. Engl. J. Med., 327: 1209-1238) and monophosphoryl lipid A adjuvant solubilized in 0.02% triethanolamine. Other useful adjuvants are, or are based on, bacterial endotoxin, lipid X, whole organisms or subcellular fractions of the bacteria Propionobacterium acnes or Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin and saponin derivatives such as QS21 (White, A. C. et al. (1991) Adv. Exp. Med. Biol., 303:207-210) which is now in use in the clinic (Helling, F et al. (1995) Cancer Res., 55:2783-2788; Davis, T A et al. (1997) Blood, 90: 509A (abstr.)), levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Examples of commercially available adjuvants include (a) Amphigen®, which is an oil-in-water adjuvant made of de-oiled lecithin dissolved in oil (see for example, U.S. Pat. No. 5,084,269 and US Pat Publication 20050058667A1 and (b) Alhydrogel®, which is an aluminum hydroxide gel. Aluminum is approved for human use. Adjuvants are available commercially from various sources, for example, Merck Adjuvant 65@ (Merck and Company, Inc., Rahway, N.J.). The immunogenic material can be adsorbed to or conjugated to beads such as latex or gold beads, ISCOMs, and the like. There is evidence that traditional formulations, such as Freund's adjuvant (both complete and incomplete) and Alum gel at least partially denature antigen resulting in the destruction or under-representation of conformational epitopes. The Ribi adjuvant system (RAS), which belongs to the monophosphoryl-lipid A (MPL) containing-adjuvants, can be used to overcome this problem. Results from several studies indicate that antigen formulated using MPL-containing adjuvants elicited antibodies that preferentially bound native rather than denatured antigen (Earl, P. L., et al., J. Virol 68:3015-3026 (1994); VanCott T. C., et al., J. Virol 71:4319-4330 (1997)).
Immunogens can also be supplemented with an immunostimulatory cytokine, lymphokine or chemokine. Exemplary cytokines include, without limitation, GM-CSF (granulocyte-macrophage colony stimulating factor), interleukin 1, interleukin 2, interleukin 12, interleukin 18 or interferon-gamma.
General methods to prepare immunogenic pharmaceutical compositions and vaccines are well known in the art (see, for example, Remington's Pharmaceutical Science; Mack Publishing Company Easton, Pa.).
b. Evaluating Immunogen-Elicited Responses
In one aspect, the present invention provides immunogens that can be used to raise antiviral neutralizing agents (e.g., antibodies and aptamers) by methods known to those of ordinary skill in the art. The antibodies raised can then be administered to a PIV-infected or non-PIV-infected subject for a variety of uses. In one embodiment involving hybridoma production, samples can be screened by a number of techniques to characterize binding to immunogens described herein. One approach involves ELISA binding to the inventive immunogens. Animals with sera samples which test positive for binding to one or more immunogens are candidates for use in MAb production. The criteria for selection of animals to be used in MAb production is based on the evidence of neutralizing antibody in the animals' sera or in the absence of neutralization, appropriate binding patterns against the desired immunogens.
Hybridoma supernatants derived from MAb production can be screened for ELISA, lysate and surface immunoprecipitation assays for binding to the desired immunogen. Samples which are positive in any of the binding assays can be screened for their ability to neutralize PIVs of interest. For example, PIV species, strains, or isolates can include lab adapted and primary virus strains, syncytium- and non-syncytium-inducing isolates, virus representing various geographic subtypes, and viral isolates which make use of the range of second receptors during virus entry. The neutralization assays employ either primary cell or cell line targets as required.
Advances in the field of immunology have allowed more thorough and sensitive evaluations of cellular responses to candidate immunogens and immunogenic compositions (e.g., vaccines) against PIVs. Such assays as intracellular staining (e.g., flow cytometry) and ELISPOT (an enzyme-linked immunosorbent assay format) allow for the detection and counting of cells producing molecules, such as antibodies and cytokines, indicative of a response to stimulation by an antigen. For example, isolation of splenocytes or peripheral blood monocyte cells (PBMCs) from an animal or human followed by in vitro challenge with an appropriately presented PIV epitope, and finally testing by ELISPOT and/or intracellular cytokine staining (ICS), can determine the potential for a cell-mediated immune response in recipients having been administered an immunogen.
In one embodiment, ELISA and Western blots are used to assess the antibody response. These methods can assess antibody binding, antibody neutralizing capability, antibody-mediated fusion inhibition, and antibody-dependent cytotoxicity. Such methods are well known in the art. For example, an MT-2 assay can be performed to measure neutralizing antibody responses. Antibody-mediated neutralization of a selected strains or isolates of a PIV can be measured in an MT-2 cell-killing assay (D. Montefiori et al., 1988, J. Clin. Microbial., 26:231-7). The inhibition of the formation of syncytia by the sera shows the activity of neutralizing antibodies present within the sera, induced by vaccination. Immunized test and control sera can be exposed to virus-infected cells (e.g., cells of the MT-2 cell line). Neutralization can be measured by any method that determines viable cells, such as staining, e.g., with Finter's neutral red. The percentage of protected cells can be determined by calculating the difference in absorption (A540) between test wells (cells and virus) and dividing this result by the difference in absorption between cell control wells (cells only) and virus control wells (virus only). Neutralizing titers can be expressed, for example, as the reciprocal of the plasma dilution required to protect at least 50% of cells from virus-induced killing.
In another embodiment, antiviral activity of neutralizing antibodies generated by the immunization with vaccine immunogens can be evaluated in both cell-cell fusion and neutralization assays. In the latter assay, a representative sample of lab adapted and primary virus isolates is used. Both assays are carried out according to known protocols as described in, for example, Wild et al. (1992) PNAS USA 89:10537-10541; Wild et al. (1994) PNAS USA 91:12676-12680; and Wild et al. (1994) PNAS USA 91:9770-9774. For example, in the neutralization assay, test sera can be incubated at a 1:10 dilution with virus for 1 hour at 37° C. At the end of this time, target cells can be added and the experiment returned to the incubator. On days 1, 3 and 5, post-infection complete media changes can be carried out. On day 7, PI culture supernatant can be harvested. Levels of virus replication can then be determined by p24 antigen capture. Levels of replication in test wells can be normalized against virus-only controls.
In still another embodiment, an assay can be used that determines an agent's ability to inhibit infectivity of PIV particles. For example, HeLa cells stably expressing human CD4 and CCR5 receptors and harboring a beta-galactosidase reporter gene driven by a tat-responsive fragment of a PIV long terminal repeat (LTR, such as HIV-2 LTR) can be infected with a PIV of various strains in the presence of a candidate neutralizing agent at varying concentrations. After incubating said cells for a specific period of time, the cells are lysed and beta-galactosidase activity is quantified. A lower beta-galactosidase activity recording indicates that the agent has an increased ability to inhibit PIV infectivity.
For any assay described herein, results from test agents can be compared against titration or positive controls (e.g., known broadly neutralizing antibodies) for normalization purposes.
c. Diagnostic, Prognostic and Therapeutic Methods
In one aspect, the present invention provides a method of preventing a subject from becoming infected with a primate immunodeficiency virus comprising administering to the subject a prophylactically effective amount of an immune response-eliciting agent described herein to thereby prevent the subject from becoming infected with the primate immunodeficiency virus.
In another aspect, the present invention provides a method for reducing the likelihood of a subject's becoming infected with a primate immunodeficiency virus comprising administering to the subject a prophylactically effective amount of an immune response-eliciting agent described herein to thereby reducing the likelihood of the subject's becoming infected with the primate immunodeficiency virus.
In still another aspect, the present invention provides a method for preventing or delaying the onset of, or slowing the rate of progression of, a primate immunodeficiency virus-related disease in a subject infected with a primate immunodeficiency virus, comprising administering to the subject a therapeutically effective amount of an immune response-eliciting agent described herein to thereby prevent or delay the onset of, or slowing the rate of progression of, the primate immunodeficiency virus-related disease in the subject.
Thus, in some embodiments, the immunogens described herein can be used to immunize a PIV-infected subject such that levels of PIV will be reduced in the subject. In other embodiments, the immunogens described herein can be employed to immunize a non-PIV-infected subject so that, following a subsequent exposure to a PIV that would normally result in a PIV infection, the level of PIV will be reduced or non-detectable using current diagnostic tests. In some embodiments, the immune response is a B cell response, a T cell response, or a combination thereof.
It will be appreciated that individual dosages can be varied depending upon the requirements of the subject in the judgment of the attending clinician, the severity of the condition being treated and the particular compound being employed. In determining the therapeutically effective amount or dose, a number of additional factors can be considered by the attending clinician, including, but not limited to: the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the desired time course of treatment; the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment; and other relevant circumstances.
Treatment can be initiated with smaller dosages which are less than the effective dose of the compound. Thereafter, in one embodiment, the dosage should be increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage can be divided and administered in portions during the day if desired.
The duration and/or dose of treatment with antiviral therapies can vary according to the particular agent or combination thereof. An appropriate treatment time for a particular antiviral therapeutic agent will be appreciated by the skilled artisan. The invention contemplates the continued assessment of optimal treatment schedules for each antiviral therapeutic agent, where the phenotype of the PIV infection of the subject as determined by the methods of the invention is a factor in determining optimal treatment doses and schedules.
In general, it is preferable to obtain a first sample from the subject prior to beginning therapy and one or more samples during treatment. In such a use, a baseline of expression of cells from subjects with a PIV infection or related disorders prior to therapy is determined and then changes in the baseline state of expression of cells from subjects with a PIV infection or related disorders is monitored during the course of therapy. Alternatively, two or more successive samples obtained during treatment can be used without the need of a pre-treatment baseline sample. In such a use, the first sample obtained from the subject is used as a baseline for determining whether the expression of cells from subjects with a PIV infection or related disorders is increasing or decreasing.
It may further be advantageous to administer the immunogenic compositions disclosed herein with other agents, such as proteins, peptides, antibodies, and other anti-PIV agents. Examples of such anti-PIV therapeutic agents include nucleoside reverse transcriptase inhibitors, such as abacavir, AZT, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zalcitabine, zidovudine, and the like, non-nucleoside reverse transcriptase inhibitors, such as delavirdine, efavirenz, nevirapine, protease inhibitors such as amprenavir, atazanavir, indinavir, lopinavir, nelfinavir osamprenavir, ritonavir, saquinavir, tipranavir, and the like, and fusion protein inhibitors such as enfuvirtide and the like. In certain embodiments, immunonogenic compositions are administered concurrently with other anti-PIV therapeutic agents. In certain embodiments, the immunonogenic compositions are administered sequentially with other anti-PIV therapeutic agents, such as before or after the other agent. One of ordinary skill in the art would know that sequential administration can mean immediately following or after an appropriate period of time, such as hours days, weeks, months, or even years later.
VI. Pharmaceutical Compositions
In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of a composition described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents and with or without additional antiviral agents and/or immunogens. As described in detail below, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
The phrase “pharmaceutically acceptable” is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
The term “pharmaceutically-acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates (e.g., enhances) envelope expression and/or activity, or expression and/or activity of the complex encompassed by the invention. These salts can be prepared in situ during the final isolation and purification of the agents, or by separately reacting a purified agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).
In other cases, the agents useful in the methods of the present invention can contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term “pharmaceutically-acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of agents that modulates (e.g., enhances) envelope expression and/or activity, or expression and/or activity of the complex. These salts can likewise be prepared in situ during the final isolation and purification of the agents, or by separately reacting the purified agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra).
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
Methods of preparing these formulations or compositions include the step of bringing into association an agent that modulates (e.g., increases or decreases) envelope expression and/or activity, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient. A compound can also be administered as a bolus, electuary or paste.
In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.
Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They can also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They can be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions, which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active agent can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Formulations for rectal or vaginal administration can be presented as a suppository, which can be prepared by mixing one or more agents with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration of an agent that modulates (e.g., increases or decreases) envelope expression and/or activity include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component can be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which can be required.
The ointments, pastes, creams and gels can contain, in addition to an agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to an agent that modulates (e.g., increases or decreases) envelope expression and/or activity, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
The agent that modulates (e.g., increases or decreases) envelope expression and/or activity, can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.
Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
Transdermal patches have the added advantage of providing controlled delivery of an agent to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the peptidomimetic in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more agents in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which can be reconstituted into sterile injectable solutions or dispersions just prior to use, which can contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of an agent that modulates (e.g., increases or decreases) envelope expression and/or activity, in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention can be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
Nucleic acid molecules described herein can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054 3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
This invention is further illustrated by the following examples, which should not be construed as limiting.
A. Protein Engineering, Expression, and Purification
The env cDNA was codon-optimised and subcloned into the pcDNA3.1(−) expression plasmid (Invitrogen). The Env(−)ΔCT glycoprotein contains a heterologous signal sequence from CD5 in place of the wild-type HIV-1 Env signal peptide. Site-directed mutagenesis was used to change the proteolytic cleavage site between gp120 and gp41, substituting Ser for Arg508 and Arg511. The Env cytoplasmic tail was truncated by introduction of a stop codon at Tyr712; a sequence encoding a (Gly)2(His)6 tag was inserted immediately before the stop codon. The plasmid expressing the Env(−)ΔCT glycoprotein was transfected into the 293F cells. After 36 h, cells expressing the envelope glycoproteins were harvested and washed with phosphate-buffered saline (PBS) at 4° C. The cell pellets were homogenized in a homogenization buffer (250 mM sucrose, 10 mM Tris-HCl [pH 7.4]) and a cocktail of protease inhibitors [Roche Complete tablets]). The plasma membranes were then extracted from the homogenates by ultracentrifugation and sucrose gradient separation. The extracted crude plasma membrane pellet was collected and solubilized in a solubilization buffer containing 100 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8), 300 mM NaCl, 20 mM imidazole, 1% (wt/vol) Cymal-5 (Affymatrix) and a cocktail of protease inhibitors (Roche Complete tablets). The membranes were solubilized by incubation at 4° C. for 30 min on a rocking platform. The suspension was ultracentrifuged for 30 min at 200,000×g at 4° C. The supernatant was collected and mixed with a small volume of pre-equilibrated Ni-NTA beads (QIAGEN) for 8-12 h on a rocking platform at 4° C. The mixture was then injected into a small column and washed with a buffer containing 100 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8), 1 M NaCl, 30 mM imidazole and 0.5% Cymal-5. The bead-filled column was eluted with a buffer containing 100 mM (NH4)2SO4, 20 mM Tris-HCl (pH 7.4), 250 mM NaCl, 250 mM imidazole and 0.5% Cymal-5. The eluted Env glycoprotein solution was concentrated, diluted in a buffer containing 20 mM Tris-HCl, pH 7.4, 300 mM NaCl and 0.01% Cymal-6, and reconcentrated to ˜2.5 mg ml-1 prior to cryo-sample preparation. The recognition of the purified Env glycoproteins by a number of conformation-dependent antibodies, including VRC01, b12 and 2G12, as well as CD4-Ig, was measured in an enzyme-linked immunosorbent assay (ELISA) (see below). The VRC01 and b12 antibodies recognize conformation-dependent epitopes near the CD4-binding site of gp120 (Zhou et al. (2007) Nature 445:732-737; Wu et al. (2010) Science 329:856-861; Zhou et al. (2010) Science 329:811-817). The 2G12 antibody recognizes a high-mannose glycan array on the gp120 outer domain (Trkola et al. (1996) J. Virol. 70:1100-1108). CD4-Ig consists of the two N-terminal domains of CD4 fused to the Fc portion of the immunoglobulin heavy chain (Finzi et al. (2010) Mol. Cell 37:656-667). Whether the Env solubilization and purification approach affected the integrity of an epitope that is recognized by the PG16 antibody and that is sensitive to changes in the quaternary structure of the HIV-1 Env trimer (Walker et al. (2009) Science 326, 285-289) was also analyzed. To this end, PG16 binding to the Env(−)ΔCT E168K glycoprotein was analyzed. The wild-type HIV-1JR-FL isolate is highly resistant to neutralization by the PG16 antibody, but the E168K change renders the HIV-1JR-FL Env sensitive to PG16. PG16 binding to the purified Env(−)ΔCT E168K glycoprotein was tested in the ELISA.
B. ELISA
A white, high-binding microtiter plate (Corning) was coated by incubating 0.5 μg of mouse anti-histidine antibody (sc-53073, Santa Cruz Biotechnology) in 100 μl PBS in each well overnight. Wells were blocked with blocking buffer (5% non-fat dry milk (Bio-Rad) in 20 mM Tris-HCl, pH 7.4 and 300 mM NaCl) for 2 hours and then washed twice with wash buffer (20 mM Tris-HCl, pH 7.4 and 300 mM NaCl). Approximately 0.5 μg of purified Env trimer in blocking buffer was added to each well, the plate was incubated for 60 minutes and washed thrice with wash buffer. Different concentrations of specific Env ligands (conformation dependent antibodies and the CD4-Ig fusion protein) in blocking buffer were added to the wells and the plate was incubated for another 45 minutes. After three washes, peroxidase-conjugated F(ab′)2 fragment donkey anti-human IgG (1:3600 dilution; Jackson ImmunoResearch Laboratories) in blocking buffer was added to each well. The plate was incubated for 30 minutes, washed six times, and 80 μl of SuperSignal chemiluminescent substrate (Pierce) was added to each well. The relative light units in each well were measured for two seconds with a Centro LB 960 luminometer (Berthold Technologies, TN). All procedures were performed at room temperature.
C. Flow Cytometry
HEK293T cells were transfected, by either calcium phosphate coprecipitation or by using the Effectene transfection reagent (Qiagen), with a plasmid encoding the Env(−)ΔCT E168K glycoproteins. After 48 hours, approximately half a million cells were analyzed by flow cytometry as previously described in Herschhorn et al. (2010) J. Immunol. 185:7623-7632, but with primary antibody incubation for 30 minutes, and secondary antibody (Allophycocyanin conjugated F(ab′)2 fragment donkey anti-human IgG antibody, Jackson ImmunoResearch Laboratories) incubation for 15 minutes, both at room temperature. Cells were analyzed with a BD FACSCanto II flow cytometer (BD Biosciences).
D. Cryo-EM Reconstruction and Model Analysis
To prepare the cryo-sample for single-particle imaging, 2.5 μl of 2.5 mg/ml Env(−)ΔCT solution was spread on a C-flat holey carbon grid (Electron Microscopy Sciences) in a chamber of 100% humidity, held for 2 seconds, blotted by filter papers for 2 seconds at 4° C., and then flash-plunged into liquid ethane by Vitrobot (FEI). The prepared cryo-grids were transferred into the CT3500 cryo-transfer system (Gatan) in liquid nitrogen and were used for single-particle image data collection at −183° C. Focus pairs of micrographs were recorded on a Tecnai F20 TEM (FEI) with a field-emission gun at 200 kV and a calibrated magnification of 200,835× on a 4 k×4 k slow-scan CCD camera (Gatan). The electron dose of each exposure was 10.0 electrons Å−2. The defocus of the second set of micrographs differed from that of the first set by 1.0 μm.
Micrographs were screened for drift, astigmatism and visibility of Thon rings in the power spectra. Parameters of the contrast transfer function (CTF) of each micrograph were determined with the CTFFind3 program (Huang et al. (2003) J. Struct. Biol. 144:79-94; Mindell & Grigorieff (2003) J. Struct. Biol. 142:334-347). A total of 90,306 single-particle images (in a dimension of 320×320 pixels and a pixel size of 0.747 Å) selected from the closer-to-focus micrographs were used for reconstruction. Each single-particle image was decimated by 4 times to a dimension of 80×80 pixels prior to further image analysis, resulting in a pixel size of 2.99 Å. The images were CTF-corrected by the phase flipping method. These single-particle images were then subjected to multivariate data analysis and classification. Images in each class were aligned in a reference-free manner and the class averages were refined by a maximum-likelihood approach (Sigworth et al. (1998) J. Struct. Biol. 122:328-339; Scheres et al. (2005) J. Mol. Biol. 348:139-149). These class averages were used to perform angular reconstitution to yield an initial model. The initial model was further refined by the projection-matching algorithm with C3 symmetry imposed. The angular increment was progressively decreased from 10° to 1° in the refinement. For the last round of refinement, the new classes of images were re-aligned by a maximum-likelihood approach (Sigworth et al. (1998) J. Struct. Biol. 122:328-339; Scheres et al. (2005) J. Mol. Biol. 348:139-149). The final reconstruction at ˜10.8 Å, measured by FSC-0.5 cutoff, was not corrected for its temperature factor.
A total of 582,914 single-particle images (with dimensions of 320×320 pixels and a pixel size of 0.747 Å) selected from the closer-to-focus micrographs (3347 in total) were used for higher resolution reconstruction. The defocus ranged from 350 to 2000 nm. The quality of these particle images was evaluated and verified comprehensively by unsupervised classification using multivariate data analysis and K-means clustering as previously described (Shaikh, et al. (2008). J. Struct. Biol. 164:41). Each single-particle image was decimated by 4 times to a dimension of 80×80 pixels, was CTF-corrected by the phase flipping method, and was low-pass filtered at 12 Å prior to particle verification and initial alignment. The initial alignment for projection Euler angles and in-plane shift was generated by a projection-matching algorithm using the previously determined 10.8-Å map described above as a reference (EMDB accession code: EMD-5418). The particles images were grouped into 57 defocus groups, with a defocus width of 20 nm in each group prior to model refinement. The refinement progressed from 4-fold decimated images, to 2-fold decimated images, and finally to non-decimated images. For the refinement with non-decimated images, the particle images were re-windowed and the dimensions reduced to 256×256 pixels without changing the pixel size (0.747 Å) in order to speed up the calculations. C3 symmetry was imposed in the refinement only with non-decimated images. The back-projection reconstruction at each iteration of the refinement was CTF-corrected by Wiener filtering (Penczek. P. A. (2010) Meth. Enzymol. 482:35 and Frank, J. Three-dimensional electron microscopy of macromolecular assemblies: visualization of biological molecules in their native state. (Oxford Univ. Press, 2006). The angular increment was progressively decreased from 10° to 0.5° in the refinement. The above image analysis was implemented in customized computational procedures and workflows, combining the functions of SPDER, XMIPP and custom-made FORTRAN programs (Shaikh et al. (2008) Nat. Protoc. 3:1941-1974; Scheres et al. (2008) Nat. Protoc. 3:977-990). The final map was deconvoluted and amplitude-corrected by a B-factor of 250 Å2 and was low-pass filtered at 5.6 Å with a cosine edge of an 8-Fourier-pixel width (Rosenthal & Henderson (2003) J. Mol. Biol. 333:721 and Fernandez, et al. (2008). J. Struct. Biol. 164:170). The resolution of the refined cryo-EM map, measured by FSC-0.5 cutoff, is 6 Å without masking the background noise in the map and is 5.66 Å with masking the background noise (Liao & Frank (2010) Structure 18:768).
E. Structure Analysis
Segmentation of the cryo-EM density was done in UCSF Chimera (Pettersen et al. (2004) J. Comput. Chem. 25:1605-1612). Flexible fitting of the crystal structure was performed by manual adjustment in O (Jones, T. A. (2004) Acta Crystallog. D 60:2115) and Coot (Emsley, et al. (2010) Acta Crytallog. D 66:486) and simulated annealing and energetic optimization in CNS (Brunger, A. T. (2007) Nat. Protoc. 2:2728) and Modeller (Marti-Renom, et al. (2000) Annu. Rev. Biophys. Biomol. Struct. 29:291). Analysis of CD4BS antibody interaction with the Env trimer was done by the structure fitting and alignment features in Coot and UCSF Chimera. Graphics were done in PyMOL (Schrodinger) and UCSF Chimera.
F. Backbone Model Building
A reference model, obtained by filtering the ˜6-Å reconstruction to 8-Å, was used to align a larger dataset of about 1-million single-particle images by projection matching. Tens of iterations of angular refinement yielded a reconstruction with an estimated resolution of ˜4 Å by Fourier Shell Correlation 0.5-cutoff. The density map allowed an initial Ca model to be traced manually in the program O (Jones, T. A. (2004) Acta Crystallogr. D 60:2115-2125). Interpretation of the Ca model was initially assisted by comparisons with crystal structures of the CD4-bound HIV-1 gp120 core, primary sequence information, secondary structure predictions by I-TASSER (Roy et al. (2010) Nat. Protoc. 5:725-738) and PHYRE (Kelley & Sternberg (2009) Nat. Protoc. 4:363-371), and known patterns of Env variation, glycosylation and disulfide bond formation. Improvement and validation of the ˜4-Å reconstruction, transformation of the Ca model into a full atomic model, and refinement of the atomic model is in progress.
The Env glycoprotein derived from a primary, neutralization-resistant (Tier 2) clade B isolate, HIV-1JR-FL, was chosen for structural analysis. The gp120-gp41 proteolytic cleavage site in the HIV-1JR-FL Env was eliminated by two single-residue changes (R508S and R511S in standard HXB2 numbering). To improve the expression level on the cell surface, the gp41 cytoplasmic tail was truncated starting from Tyr712. The modified Env, designated Env(−)ΔCT, thus contains the complete ectodomain and transmembrane regions, and was purified from the plasma membrane of Env-expressing cells after solubilization in Cymal-5 detergent (see Example 1). This procedure ensured that the purified Env(−)ΔCT trimers derived from membrane-bound Env complexes that are glycosylated and have passed the quality-control checkpoints of the secretory pathway (Moulard & Decroly (2000) Biochim. Biophys. Acta 1469:121-132; Wyatt and Sodroski (1998) Science 280:1884-1888). Importantly, HIV-1 Env(−)ΔCT complexes purified in this manner retain epitopes that are dependent upon conformation, glycosylation and quaternary structure (
The HIV-1JR-FL Env(−)ΔCT complex resembles a triangular pyramid, with the transmembrane helices at the apex (
The gp41 transmembrane region anchors the Env spike to the viral membrane and, as a pivot point for the three protomeric arms, contributes to trimer-stabilizing interactions. The membrane-spanning segment of each protomer is consistent with an α-helix. The three α-helices form a left-handed coiled coil, with a crossing angle of ˜35° (
Three layers of thin curved sheets in the cryo-EM density encircle the transmembrane helices (
The transmembrane α-helical coiled coil extends beyond the boundary of the viral membrane into the membrane-proximal external region (MPER). Near the surface of the viral membrane, transverse segments of structure, likely composed of short α-helices and loops emanating from and returning to the gp41 ectodomain, wedge into the transmembrane helical bundle close to the trimer axis. Some of the density in this region exhibits features typical of glycans (see below), and may represent gp41 carbohydrate chains (
The resulting structure not only stabilizes the transmembrane helical bundle, but also creates a sufficiently wide pitch and appropriate crossing angle between the three interacting α-helices so that the gp41 ectodomain can be built upon the transmembrane helices and achieve its torus-like topology (Mao, et al. (2012) Nat. Struct. Mol. Biol. in press).
With an overall globular shape, the gp41 ectodomain from each protomer comprises at least seven major α-helical elements (
The organization of secondary structure elements in the gp41 ectodomain in this unliganded and uncleaved state of Env differs dramatically from that of the six-helix bundle structure seen in the post-fusion state (Weissenhorn, et al. (1997) Nature 387:426; Chan, et al. (1997) Cell 89:263 and Buzon, V. (2010) PLoS Pathog. 6:e100088012). In the six-helix bundle, three heptad repeat 1 (HR1) regions (residues ˜546-581) assemble into a trimeric coiled coil and three HR2 regions (residues ˜628-661) form long α-helices that pack in an antiparallel fashion into the hydrophobic grooves of the coiled coil (Pancera, et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:1166; Kwon, et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109:5663 and Weissenhorn, et al. (1997) Nature 387:426). In the unliganded state of Env, the long HR1 and HR2 α-helices in the six-helix bundle appear to be broken into short helices, consistent with previous studies suggesting that the trimeric coiled coil is not formed prior to CD4 engagement (Mische, et al. (2005) Virology 338:133 and Dimitrov, et al. (2005) Biochemistry 44:12471). The available evidence supports a model in which: 1) α-helical elements from the HR1 region locate at the membrane-distal end of the ectodomain and interact with the gp120 subunit; and 2) α-helical elements from the HR2 region locate around the membrane-proximal end of the ectodomain, contribute to the gp41 trimeric interactions and constitute the torus-shaped structure. Consistent with this model, mutagenesis experiments implicate the HR1 region in the non-covalent association with gp120 (Sen, et al. (2010) Biochemistry 49:5057) and the HR2 region in trimerization of the Env ectodomain (Helseth, et al. (1991) J. Virol. 65:2119).
The CD4-bound gp120 core consists of an inner and outer domain, as well as a bridging sheet (Kwong, et al. (1998) Nature 393:648; Zhou, et al. (2007) Nature 445:732; Pancera, et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:1166 and Kwon, et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109:5663). The full-length unliganded gp120 subunit shown in our cryo-EM map exhibits a conformation different from that observed in the CD4-bound gp120 core (Kwong, et al. (1998) Nature 393:648; Zhou, et al. (2007) Nature 445:732; Pancera, et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:1166 and Kwon, et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109:5663). Such a difference was already apparent in the previous 11-Å Env(−)ΔCT map (Mische, et al. (2005) Virology 338:133). However, the improved resolution of the current map allows positioning of the secondary structural elements in gp120 and a detailed assessment of the CD4-induced conformational change (Sattentau & Moore (1991) J. Exp. Med. 174:407; Myszka, et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:9026; Xiang, et al. (2002) J. Virol. 76:9888 and Kong, et al. (2010) J. Virol. 84:10311). The crystal structure of the monomeric gp120 core was flexibly fitted in the CD4-bound state to the cryo-EM map. Most of the secondary structures in the CD4-bound gp120 core were able to be retained in our cryo-EM structure of the unliganded trimer. Notably, the majority of the gp120 outer domain structure fit into the cryo-EM density as a rigid-body, requiring little conformational change (
Studies on monomeric gp120 suggested that the bridging sheet forms from two elements (β2/β3 and β20/β21) only after CD4 binding (Guttman, et al. (2012) J. Virol. 10.1128/JVI.07224-11 and other references). Indeed, flexible fitting of the CD4-bound gp120 core structure to the unliganded Env trimer map failed to maintain the bridging sheet in its CD4-bound conformation (Kwong, et al. (1998) Nature 393:648; Zhou, et al. (2007) Nature 445:732; Pancera, et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:1166 and Kwon, et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109:5663;
The conformations of the gp120 inner domain and bridging sheet, but not that of the outer domain, in the crystal structure of an unliganded simian immunodeficiency virus (SIV) gp120 core (Chen, et al. (2005) Nature 433:834) are incompatible with our HIV-1 Env trimer map and with the results of previous studies (Liu, et al. (2008) Nature 455:109; White, et al. (2010) PLoS Pathog. 6:e1001249; Wu, et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:18844; Hu, et al. (2011) J. Virol. 85:2741; Mao, et al. (2012) Nat. Struct. Mol. Biol. in press and Guttman, et al. (2012) J. Virol. 10.1128/JVL.07224-11). These observations make it unlikely that the unliganded SIV gp120 core structure (Chen, et al. (2005) Nature 433:834) represents the actual gp120 conformation in the unliganded HIV-1 Env trimer.
Previous studies have suggested that the gp120 V1/V2 and V3 regions (Hu, et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 107:18844 and Mao, et al. (2012) Nat. Struct. Mol. Biol. in press) are located at the membrane-distal apex of the Env spike and contribute to the association of gp120 with the trimer (Xiang, et al. (2010) J. Virol. 84:3147; Musich, et al. (2011) J. Virol. 85:2397 and Kolchinsky, et al. (2001) J. Virol. 75:3435). The flexible fitting of the gp120 core structure in our cryo-EM map demarcates the boundary of the gp120 trimer association domain (TAD) that comprises the V1/V2 and V3 regions (Mao, et al. (2012) Nat. Struct. Mol. Biol. in press). The TAD in each protomer exhibits a β-α-β architecture and extends transversely from the gp120 inner domain to the trimer axis (
Broadly neutralizing antibodies like PG9 and PG16 that recognize glycan-dependent TAD epitopes exhibit a strong preference for Env trimers (Walker, et al. (2009) Science 326:285), consistent with the complex architecture of this region. When removed from the context of the Env trimer, significant portions of the V1/V2 and V3 regions become disordered (Guttman, et al. (2012) J. Virol. 10.1128/JVI.07224-11) and recognition by the PG9 and PG16 antibodies is markedly diminished (Walker, et al. (2009) Science 326:285). By expressing the V1/V2 region fused with a heterologous scaffold protein and using an elegant strategy to select complexes with PG9, a crystal structure was obtained (McLellan, et al. (2011) Nature 480:336). Although we can fit the two V1/V2 β-strands in immediate contact with the PG9 antibody into the Env trimer map, the complete V1/V2 region from this crystal structure cannot be accommodated in either the 6-Å or the 11-Å trimer map (Mao, et al. (2012) Nat. Struct. Mol. Biol. in press). A considerable portion of the V1/V2 region remains disordered and is not resolved in the crystal structure. These regions may fold into distinct structures in the presence of other elements of the TAD, interprotomer interactions on the trimer, or contacts with the inner domain.
Flexible fitting of the gp120 core structure into the cryo-EM map revealed a number of outward-protruding densities on the surface of the gp120 outer domain that cannot be attributed to protein (
The CD4-binding site (CD4BS) antibodies are directed against the conserved gp120 surface that engages CD4, are elicited in some HIV-1-infected individuals and exhibit a range of potencies of HIV-1 neutralization (Chen, et al. (2009) Science 326:1123-1127). What accounts for these differences in potency? Crystal structures of several CD4BS antibodies complexed with the HIV-1 gp120 core are available (Zhou et al. (2010) Science 329:811-815; Chen, et al. (2009) Science 326:1123-1127). The overall conformation of the gp120 outer domain in the unliganded HIV-1 Env trimer does not significantly differ from that in the crystal structures of gp120 cores complexed with CD4 or CD4BS antibodies. As all CD4BS antibodies include the gp120 outer domain elements in their epitopes, we could superpose the crystal structures of the antibody-bound gp120 core onto our Env trimer structure by aligning the outer domains. This allows an assessment of the angle-of-approach taken by the CD4BS antibodies and CD4 as they initially engage the unliganded Env trimer, and reveals remarkable differences among CD4BS antibodies (
The Calpha model of the HIV-1JR-FL Env(−)ΔCT trimer is shown in
Enveloped viruses employ conformational changes in their envelope proteins to mediate the fusion of viral and target cell membranes. Environmental triggers such as endocytosis/pH decrease or receptor binding drive the envelope proteins to energetically favorable, fusion-ready conformations. Influenza virus uses low pH, which globally alters the conformation of the hemagglutinin (HA) protein, as a trigger. A spring-loaded mechanism has been suggested to explain the HA conformational change that mediates virus entry (Wilson, et al. (1981) Nature 289:366 and Carr & Kim (1993) Cell 73:823). The long α-helix in the fusion-ready HA2 protein is broken into several shorter pieces in the prefusion state, resembling a “loaded spring”. When triggered by a pH decrease, the HA2 short helices and linking loops refold into a longer helix of lower free energy. This allosteric change delivers the fusion peptide at the HA2 N-terminus to the target membrane and promotes virus entry.
Instead of a decrease in pH, HIV-1 utilizes sequential binding to the CD4 and CCR5 receptors as triggers for entry-related conformational changes in Env. The unliganded Env trimer is prestressed, storing all of the energy needed for membrane fusion in its unique architecture. Spring-loading appears to be used twice in the unliganded gp41 subunit, i.e., the HR1 and HR2 helices of the post-fusion six-helix bundle are each broken into smaller helices and other structural elements in the membrane-distal gp120-gp41 interaction interface and in the membrane-proximal torus, respectively. This dual spring-loaded mechanism likely reflects the unique requirements of triggering by dual receptor engagement, so that binding to each receptor frees only one “spring” at a time. Consistent with this model, experimental observations suggest that CD4 binding induces the formation/exposure of the HR1 coiled coil, but subsequent events such as CCR5 binding are required for formation of HR2 and the six-helix bundle (Futura et al. (1998) Nat. Struct. Biol. 5:276-281).
Achieving a prestressed structure that implements a dual spring-loaded mechanism imposes significant challenges to folding; indeed, HIV-1 Env synthesis and assembly is relatively slow and inefficient. Once synthesized, functional Env spikes must avoid premature triggering. Synthesis and maintenance of a competent, spring-loaded Env trimer requires multiple interactions between and within the protomers. The elegant torus-like architecture of gp41 takes advantage of the high stability and pivot potential of the transmembrane three-helix bundle. Truncations of the gp41 transmembrane region to produce “soluble gp140” trimers (Binley, et al. (2000) J. Virol. 74:627) typically disrupt the native Env structure, as indicated by loss of PG9 and PG16 epitopes (McLellan, et al. (2011) Nature 480:336). Packing of the short gp41 helices and interactions with the gp120 inner domain and N/C termini likely maintain the spring-loaded conformation of the membrane-distal gp41 ectodomain. Interactions between the TAD and the gp120 inner domain layers and bridging sheet components explain the ability of the V1/V2 and V3 regions to prevent gp120 from assuming the energetically favored CD4-bound state (Kwon, et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109:5663). Conversely, CD4 binding necessitates TAD restructuring, thus decreasing gp120 trimerization and allowing an open conformation of the trimer (Liu, et al. (2008) Nature 455:109). The “spring-loaded” architecture of the HIV-1 Env is well adapted to respond to the relatively subtle perturbation of initial receptor engagement with dramatic, programmed conformational changes. Sensitive adjustment of local features allows HIV-1 variants to adapt to differing levels of receptors and to escape antibody neutralization. The ability of the HIV-1 Env to fuse membranes at neutral pH contributes to cytopathic events in infected lymphocytes and to loss of immunocompetence in the infected host (Etemad-Moghadam, et al. (2001) J. Virol. 75:5646). Thus, the unique architecture of Env trimer offers multiple fitness advantages to a persistent virus like HIV-1.
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The contents of all references, patent applications, patents, and published patent applications, as well as the Figures and the Sequence Listing, cited throughout this application are hereby incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application is the U.S. national phase of International Patent Application No. PCT/US2013/0052855, filed on 31 Jul. 2013, which claims the benefit of priority to U.S. Provisional Patent Application No. 61/742,139 filed Aug. 3, 2012, the entire content of each application is incorporated herein in their entirety by this reference.
This invention was made with government support under grant numbers AI093256, AI067854, and AI024755 awarded by The National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/052855 | 7/31/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/022475 | 2/6/2014 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 7939083 | Dey | May 2011 | B2 |
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