Patent Application
20160235836
This disclosure relates to immunogenic polypeptides, particularly polypeptides that can elicit an immune response to human immunodeficiency virus (HIV) in a subject.
Approximately 35 million people worldwide are estimated to be infected with human immunodeficiency virus (HIV). Although infection rates are declining, in 2012 about 2.3 million people were newly infected and about 1.6 million people died from AIDS-related illnesses. However, viral diversity in HIV and the occurrence of escape variants provide significant challenges to development of effective HIV vaccines. Thus, there remains a need for the development of effective vaccines to treat and inhibit HIV infection worldwide.
Disclosed herein are mosaic HIV envelope (Env) proteins that can elicit an immune response to HIV (such as cytotoxic T cell (CTL), helper T cell, and/or humoral responses). In specific examples, the disclosed mosaic proteins (also referred to herein as mosaic Env proteins or immunogenic polypeptides) can elicit B cell responses to HIV. Also disclosed are sets of mosaic Env proteins, which include two or more (for example, three or more) of the proteins. In some embodiments, the disclosed mosaic Env proteins or sets of mosaic Env proteins are included in an immunogenic composition, such as a polyvalent immunogenic composition.
Also disclosed herein are methods for treating or inhibiting HIV in a subject including administering one or more of the disclosed immunogenic polypeptides or compositions to a subject infected with HIV or at risk of HIV infection. In some embodiments, the methods include inducing an immune response to HIV in a subject, comprising administering to the subject at least one (such as two, three, or more) of the disclosed immunogenic polypeptides or at least one (such as two, three, or more) nucleic acids encoding at least one of the immunogenic polypeptides disclosed herein.
The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
The nucleic acid and amino acid sequences disclosed herein and in the accompanying Sequence Listing are shown using standard letter abbreviations for nucleotide bases, and one letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
SEQ ID NOs: 1-8 are the amino acid sequence of exemplary Env mosaic proteins.
SEQ ID NO: 9 is the amino acid sequence of a tissue plasminogen activator leader peptide.
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.
As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes single or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” Thus, “comprising an antigen” means “including an antigen” without excluding other elements.
It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
To facilitate review of the various embodiments of the disclosure, the following explanations of terms are provided:
Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example see U.S. Pat. No. 6,194,388; U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,214,806; U.S. Pat. No. 6,218,371; U.S. Pat. No. 6,239,116; U.S. Pat. No. 6,339,068; U.S. Pat. No. 6,406,705; and U.S. Pat. No. 6,429,199). Adjuvants include biological molecules (a “biological adjuvant”), such as costimulatory molecules. Exemplary biological adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL. Adjuvants can be used in combination with the disclosed immunogenic polypeptides.
Administration: The introduction of a composition into a subject by a chosen route. For example, if the chosen route is intravenous, the composition (such as a disclosed antigen) is administered by introducing the composition intravenously into a subject.
Antibody: A polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an analyte (such as an antigen or immunogen) such as a Env polypeptide or antigenic fragment thereof. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
Antibodies exist, for example as intact immunoglobulins and as a number of well characterized fragments produced by digestion with various peptidases. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to Env would be Env-specific binding agents. This includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab′ fragments, F(ab)′2 fragments, single chain Fv proteins (scFv), and disulfide stabilized Fv proteins (dsFv). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term antibody also includes genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.
Antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab′)2, a dimer of two Fab′ fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody (SCA), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. The term “antibody” as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.
Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs.” The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes referred to as CDR L1, CDR L2, and CDR L3. Heavy chain CDRs are sometimes referred to as CDR H1, CDR H2, and CDR H3.
References to “VH” or “VH” refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab. References to “VL” or “VL” refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
A “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed “hybridomas.” Monoclonal antibodies include humanized monoclonal antibodies.
Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in a subject, including compositions that are injected or absorbed into a subject. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. “Epitope” or “antigenic determinant” refers to the region of an antigen to which B and/or T cells respond. In one embodiment, T cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, about 8-10, or about 6-22 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and nuclear magnetic resonance.
Examples of antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids containing antigenic determinants, such as those recognized by an immune cell. In some examples, antigens include peptides derived from a pathogen of interest. Exemplary pathogens include bacteria, fungi, viruses and parasites. In specific examples, an antigen is derived from HIV, for example, one or more HIV polypeptides or a fragment thereof, such as at least a portion of an Env protein.
B cell: A lymphocyte, a type of white blood cell that expresses immunoglobulin on its surface and can ultimately develop into an antibody secreting a plasma cell. In one example, a B cell expresses CD19 (CD19+). An “immature B cell” is a cell that can develop into a mature B cell. Generally, pro-B cells (that express, for example, CD45 or B220) undergo immunoglobulin heavy chain rearrangement to become pro-B pre-B cells, and further undergo immunoglobulin light chain rearrangement to become an immature B cells. Immature B cells include T1 and T2 B cells. Immature B cells express IgM on their cell surface and can develop into mature B cells, which can express different forms of immunoglobulin (e.g., IgA, IgG). B cells can be activated by agents such as lipopolysaccharide (LPS), CD40 ligation, and antibodies that crosslink the B cell receptor (immunoglobulin), including antigen, or anti-Ig antibodies. Neutralizing antibodies can inhibit HIV infection of the natural viral target cell, CD4 positive T cells.
Envelope (Env): The envelope protein from HIV. Env is initially synthesized as a precursor protein of 845-870 amino acids in size (gp160). gp160 forms a homotrimer and undergoes glycosylation in the Golgi apparatus. It is then cleaved by a cellular protease into gp120 and gp41. gp120 includes most of the surface-exposed domains of the Env glycoprotein complex and binds to the cellular CD4 receptor and cellular chemokine receptors (for example, CCR5). Env is the only HIV protein capable of stimulating HIV neutralizing antibodies.
HXB2 numbering system: A reference numbering system for HIV protein and nucleic acid sequences, which uses HIV-1 HXB2 strain sequences as a reference for all other HIV strain sequences. The person of ordinary skill in the art is familiar with the HXB2 numbering system (Korber et al., Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken C L, Foley B, Hahn B, McCutchan F, Mellors J W, and Sodroski J, Eds.; Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N.M., incorporated by reference herein in its entirety). HXB2 is also known as: HXBc2, for HXB clone 2; HXB2R, in the Los Alamos HIV database, with the R for revised, as it was slightly revised relative to the original HXB2 sequence; and HXB2CG in the NCBI GenBank database, for HXB2 complete genome.
Host cells: Cells in which a virus or vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.
Immunogenic polypeptide: A protein or a portion thereof that is capable of inducing an immune response in a subject, such as a subject infected with, or at risk of infection with, a pathogen. Administration of an immunogenic polypeptide derived from a pathogen of interest can elicit an immune response against the pathogen. Administration of an immunogenic polypeptide can lead to protective immunity against a pathogen of interest (such as HIV). In some examples, an immunogenic polypeptide is a polypeptide including one or more regions from an HIV proteome, for example, an Env protein, such as a mosaic Env protein.
Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an “antigen-specific response”). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies. Some HIV neutralizing antibody responses can be type-specific, and only elicit responses that neutralize a single strain, while others are broad and can elicit responses to many different strains.
Immunogenic composition: A composition comprising an immunogenic polypeptide or a nucleic acid encoding an immunogenic polypeptide that induces a measurable CTL response against virus expressing the immunogenic polypeptide or a portion thereof, induces a measurable helper T cell response, or induces a measurable B cell response (such as production of antibodies) against the immunogenic polypeptide or a portion thereof. In one example, an “immunogenic composition” is composition including one or more polypeptides from HIV, such as one or more of the mosaic Env proteins disclosed herein. It further refers to isolated nucleic acids encoding an immunogenic polypeptide, such as a nucleic acid that can be used to express the immunogenic polypeptide (and thus be used to elicit an immune response against this polypeptide or a portion thereof).
For in vitro use, an immunogenic composition may consist of at least one (such as two or more) isolated polypeptides, peptide epitopes, or nucleic acids encoding the polypeptide or peptide epitope. For in vivo use, the immunogenic composition will typically include at least one (such as one, two, three, or more) polypeptide, peptide, or nucleic acid in pharmaceutically acceptable carriers, and/or other agents. Any particular peptide, such as a disclosed polypeptide or a nucleic acid encoding the polypeptide, can be readily tested for its ability to induce a CTL, helper T cell, or B cell response by art-recognized assays. Immunogenic compositions can include adjuvants, which are well known to one of skill in the art.
Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as acquired immune deficiency syndrome (AIDS), AIDS-related conditions, HW infection (such as HIV-1 infection), or combinations thereof. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition (such as AIDS or AIDS related conditions) after it has begun to develop. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
Isolated: An “isolated” biological component (such as a protein, for example a disclosed polypeptide or nucleic acid encoding such a polypeptide) has been substantially separated or purified away from other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, and nucleic acids that have been “isolated” include proteins purified by standard purification methods. The term also embraces proteins or peptides prepared by recombinant expression in a host cell as well as chemically synthesized proteins, peptides, and nucleic acid molecules.
Isolated does not require absolute purity, and can include protein, peptide, or nucleic acid molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.
Mosaic polypeptide or mosaic protein: A polypeptide or protein assembled from fragments of natural sequences via computational optimization (e.g., Fischer et al., Nat. Med. 13:100-106, 2007). Multiple sequences (for example, thousands of sequences) are used as input and the sequences are evolved by recombination in silico. Recombinants are constrained to have natural breakpoints and a mosaic set is designed to maximize coverage of potential epitopes (such as B cell epitopes) for a viral population.
Operably linked: A first nucleic acid is operably linked with a second nucleic acid when the first nucleic acid is placed in a functional relationship with the second nucleic acid. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acids are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. In some examples, the operably linked nucleic acids are heterologous, for example, the first and second nucleic acids are from different organisms, different genes, or different polypeptides and the resulting nucleic acid is not naturally occurring.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the proteins, nucleic acids, and other compositions herein disclosed.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions, powder, pill, tablet, or capsule forms, conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Polypeptide: Any compound composed of amino acids and/or amino acid analogs, chemically bound together. Polypeptide as used herein includes oligomers of amino acids and/or amino acid analogs, or small and large peptides, including proteins. Any chain of amino acids, regardless of length or post-translational modification (such as glycosylation or phosphorylation) is referred to as a polypeptide. The term polypeptide applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymers as well as polymers in which one or more amino acid residue is a non-natural amino acid, for example an artificial chemical mimetic of a corresponding naturally occurring amino acid. As used herein, polypeptide also refers to recombinant amino acid polymers, such as polymers including portions that are obtained from different (typically non-contiguous) portions of a genome (such as an HIV genome) and/or are obtained from different genomes (such as two or more HIV strains). A “residue” refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic.
Polyvalent immunogenic composition: A composition including two or more separate immunogenic polypeptides (such as a “cocktail” of immunogenic polypeptides) that are capable of eliciting an immune response in a subject, for example an immune response to HIV. In some examples, a polyvalent immunogenic composition includes two or more immunogenic polypeptides (or nucleic acids encoding the polypeptides). In one specific example, a polyvalent immunogenic composition includes three Env proteins, such as three Env mosaic proteins disclosed herein.
Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein is one in which the protein is more enriched than the protein is in its natural environment within a cell. Preferably, a preparation is purified such that the protein represents at least 50% of the protein content of the preparation.
Recombinant nucleic acid or polypeptide: A nucleic acid molecule or polypeptide that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of nucleotide or amino acid sequence. This artificial combination is accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The term “recombinant” includes nucleic acids or polypeptides that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule or peptide.
Sequence identity/similarity: Sequence identity between two or more nucleic acid sequences or between two or more amino acid sequences can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.
In some examples, sequence similarity is assessed by the conservation of epitope-length fragments. The use of this measure of similarity was developed at Los Alamos National Laboratory, and tools are available on the World Wide Web at hiv.lanl.gov.
Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals (including non-human primates).
Therapeutically effective amount or effective amount: The amount of an agent, such as a nucleic acid, polypeptide, or other therapeutic agent, that is sufficient to prevent, treat (including prophylaxis), reduce and/or ameliorate the symptoms and/or underlying causes of a disorder or disease, for example to prevent, inhibit, and/or treat HIV. In some embodiments, an “effective amount” is sufficient to reduce or eliminate a symptom of a disease, such as AIDS. For instance, this can be the amount necessary to inhibit viral replication or to measurably alter outward symptoms of the viral infection, such as an increase of T cell counts in the case of an HIV infection. In general, this amount will be sufficient to measurably inhibit virus (for example, HIV) replication or infectivity. An “anti-viral agent” or “anti-viral drug” is an agent that specifically inhibits a virus from replicating or infecting cells. Similarly, an “anti-retroviral agent” is an agent that specifically inhibits a retrovirus from replicating or infecting cells.
Transformed: A transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.
Vaccine: A pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response and can block subsequent infection, in other cases it can limit the pathological impact of an infection by containing the infection. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a pathological condition. A vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell, or one or more cellular constituents.
Vector: A nucleic acid molecule that can be introduced into a host cell, thereby producing a transformed host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.
Virus: A virus consists essentially of a core of nucleic acid surrounded by a protein coat, and has the ability to replicate only inside a living cell. “Viral replication” is the production of additional virus by the occurrence of at least one viral life cycle. A virus may subvert the host cell's normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so. In some examples, a virus is a pathogen.
“Retroviruses” are RNA viruses wherein the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a provirus. The term “lentivirus” is used in its conventional sense to describe a genus of viruses containing reverse transcriptase. The lentiviruses include the “immunodeficiency viruses” which include human immunodeficiency virus (HIV) type 1 and type 2 (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV).
HIV-1 is a retrovirus that causes immunosuppression in humans (HIV disease), and leads to a disease complex known as the acquired immunodeficiency syndrome (AIDS). “HIV disease” refers to a well-recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by an HIV virus, as determined by antibody or western blot studies or detection of HIV nucleic acids. Laboratory findings associated with this disease are a progressive decline in T cells.
Disclosed herein are mosaic polypeptides from HIV Env protein (also referred to herein as immunogenic polypeptides). The mosaic polypeptides (also referred to as mosaic proteins) are computationally designed to optimally cover global HIV diversity (e.g., having the potential to elicit broadly cross-reactive immune responses), as described in Examples 1 and 2, below. Mosaic polypeptides are assembled from fragments of natural sequences via a computational optimization method (e.g., U.S. Pat. App. Publ. No. 2012/0231028, incorporated herein by reference in its entirety). In some embodiments, mosaic polypeptides resemble natural proteins, but do not exist in nature. Thousands of sequences are use used as input, and the sequences are evolved by recombination in silico. Recombinants are constrained to have natural breakpoints, and a mosaic set will maximize the coverage of potential epitopes (such as B cell epitopes) for a viral population. Combinations of mosaics are selected to give the optimal coverage of potential epitopes found in natural sequences for a given number of mosaics. The Env mosaic proteins and nucleic acids encoding the proteins disclosed herein are capable of eliciting an immune response to HIV in a subject.
Also disclosed herein are sets of the immunogenic polypeptides (such as sets of two or more polypeptides) that can be used to elicit an immune response to HIV in a subject. B cells undergo a process called affinity maturation, where they evolve to improve affinity for their target epitope during the immune response. Without being bound by theory, it is believed that by administering a set of Env polypeptides including coverage of multiple HIV variants to a subject, exposing a B cell lineage to common variants of an HIV epitope during affinity maturation may yield an antibody response with greater breadth and ability to interact with natural variants, by selecting for antibodies that high affinity for the most common forms of the epitope.
A. Immunogenic Polypeptides
Exemplary amino acid sequences of HIV Env mosaic proteins identified as described in Example 2 include SEQ ID NOs: 1-8 disclosed herein. In some examples, the disclosed polypeptides include, consist essentially of, or consist of an amino acid sequence at least 95% identical to the amino acid sequence set forth as one of SEQ ID NOs: 1-8, such as at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or even 100% identical to the sequence set forth as one of SEQ ID NOs: 1-8.
In some embodiments, the disclosed Env mosaic proteins are utilized in combination, for example as sets of immunogenic polypeptides. In some examples, the set of Env mosaic proteins includes 2, 3, 4, 5, 6, 7, or 8 of the disclosed polypeptides. The sets of polypeptides are selected for providing coverage of variants within a single HIV clade (for example, within clade C), providing coverage of variants between clades (for example, at least clade B, clade C, and CRF01), and/or global coverage (for example M group), and can be administered to a subject, for example as a polyvalent immunogenic composition. Exemplary sets of polypeptides are shown in Table 1. However, additional combinations of the disclosed immunogenic polypeptides can also be selected to produce additional sets.
In some examples, a leader or signal peptide is linked to the immunogenic polypeptide, for example to increase expression and/or immunogenicity of the polypeptide. In one example, the leader peptide is a tissue plasminogen activator (tPA) leader peptide, for example, a peptide having the amino acid sequence MDAMKRGLCCVLLLCGAVFVSAR (SEQ ID NO: 9). One of skill in the art can identify other suitable leader peptides that can be used to optimize expression of the disclosed polypeptides.
The immunogenic polypeptides disclosed herein can be chemically synthesized by standard methods, or can be produced recombinantly, for example by expression of the polypeptide from a nucleic acid molecule that encodes the polypeptide. An exemplary process for polypeptide production is described in Lu et al., FEBS Lett. 429:31-35, 1998. They can also be isolated by methods including preparative chromatography and immunological separations.
B. Nucleic Acids
Nucleic acids encoding the disclosed Env mosaic proteins (e.g., SEQ ID NOs: 1-8) are also disclosed herein. Unless otherwise specified, a “nucleic acid encoding a polypeptide” includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. For example, a polynucleotide encoding a disclosed immunogenic polypeptide includes a nucleic acid sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide encoded by the nucleotide sequence is unchanged. In some embodiments, the disclosed polypeptide sequences are back-translated to codon optimized DNA using standard methods.
The nucleic acids encoding a disclosed polypeptide include a recombinant DNA which is incorporated into a vector, such as an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences. Methods for the manipulation and insertion of the nucleic acids of this disclosure into vectors are well known in the art (see for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y., 1994). DNA sequences encoding the polypeptide can be expressed in vitro or in vivo by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Polynucleotide sequences encoding the disclosed polypeptides can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is joined such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archaea, insect, fungi (for example, yeast), plant, and animal cells (for example, mammalian cells, such as human). Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines. Techniques for the propagation of mammalian cells in culture are well-known (see, Jakoby and Pastan (eds), 1979, Cell Culture, Methods in Enzymology, volume 58, Academic Press, Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly used mammalian host cell lines are VERO and HeLa cells, CHO cells, HEK 293 cells, and WI38, BHK, and COS cell lines, although other cell lines may be used, such as cells designed to provide higher expression, desirable glycosylation patterns, or other features.
A number of viral vectors have been constructed, that can be used to express the disclosed polypeptides, including polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol., 73:1533-1536); adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256); non-replicating adenoviruses of chimpanzee origin (ChAdv; Tatsis et al., Gene Ther. 13:421-429, 2006); vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499); modified vaccinia Ankara (MVA) virus (Kremer et al., Methods Mol. Biol. 890:59-92, 2012); adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol., 158:91-123; On et al., 1990, Gene, 89:279-282); herpes viruses, including HSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol., 66:2952-2965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199); Sindbis viruses (Herweijer et al., 1995, Human Gene Therapy 6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879); alphaviruses (Schlesinger, 1993, Trends Biotechnol. 11:18-22; Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377); and retroviruses of avian (Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol., 54:401-407), and human origin (Page et al., 1990, J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739). Baculovirus (Autographa californica multinuclear polyhedrosis virus; AcMNPV) vectors are also known in the art, and may be obtained from commercial sources (such as PharMingen, San Diego, Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).
The immunogenic polypeptides disclosed herein (such as SEQ ID NOs: 1-8, or polypeptides having at least 95% sequence identity to SEQ ID NOs: 1-8), or nucleic acids encoding the disclosed immunogenic polypeptides, can be administered to a subject to elicit an immune response in the subject, such as an immune response to HIV. In some embodiments, one or more of the disclosed polypeptides (or one or more nucleic acids encoding the disclosed polypeptides) is administered to a subject with HIV infection or at risk of HIV infection. In other embodiments, the one or more immunogenic polypeptides are administered to a subject as part of an immunization regimen. The one or more immunogenic polypeptides are administered in an amount sufficient to elicit an immune response to HIV in the subject. In some examples, administration of the immunogenic peptide inhibits (or in some instances even prevents) infection with HIV and/or reduces the signs and symptoms of HIV in an infected subject.
In particular embodiments, two or more of the disclosed polypeptides or nucleic acids encoding the polypeptides are administered to the subject. In some examples, the methods include administering to the subject one or more Env mosaic proteins (or nucleic acids encoding at least two polypeptides), for example, as a polyvalent immunogenic composition. In particular examples, the methods include administering to the subject one or more of the Env mosaic proteins (for example, 2, 3, 4, 5, 6, 7, or more Env mosaic proteins) disclosed herein.
In some embodiments, a subject is administered a set of immunogenic polypeptides, such as a set of three immunogenic polypeptides disclosed herein. In one example, a set of immunogenic polypeptides administered to the subject includes three polypeptides comprising, consisting essentially of, or consisting of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In another example, a set of immunogenic polypeptides administered to the subject includes three polypeptides comprising, consisting essentially of, or consisting of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 5. In a further example, a set of immunogenic polypeptides administered to the subject includes three polypeptides comprising, consisting essentially of, or consisting of the amino acid sequences set forth as SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In additional examples, the C clade mosaic proteins disclosed herein could be used singly as SEQ ID NO: 1; as a pair of SEQ ID NO: 1 and SEQ ID NO: 2; or as a combination of all three proteins (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3) in a polyvalent vaccine. In further examples, SEQ ID NO: 4 or SEQ ID NO. 5 could be utilized singly or as a pair (for example in geographic regions where the B clade or CRF01 dominate the regional epidemic), as well as the three protein combination of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 5. One of skill in the art can identify additional combinations of the disclosed polypeptides that could be administered to a subject as a set. In addition, the disclosed mosaic Env proteins can be combined with natural strains to form additional sets.
In some examples, the two or more immunogenic polypeptides (such as a set of immunogenic polypeptides) are administered simultaneously (for example, as a mixture), substantially simultaneously (for example, within a few minutes of one another, such as within less than 5 minutes of one another), or sequentially (for example, within 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 12 hours, 24 hours, or more of one another).
One or more of the disclosed polypeptides or nucleic acids encoding the polypeptides (including vectors including the nucleic acid) can be administered by any means known to one of skill in the art (see Banga, “Parenteral Controlled Delivery of Therapeutic Peptides and Proteins,” in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995) either locally or systemically, such as by intramuscular, subcutaneous, or intravenous injection, but even oral, nasal, or anal administration is contemplated. In one embodiment, administration is by subcutaneous or intramuscular injection. To extend the time during which the disclosed polypeptides are available to stimulate a response, the polypeptide or nucleic acid encoding the polypeptide can be provided as an implant, an oily injection, or as a particulate system. The particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle. (see, e.g., Banga, supra). A particulate carrier based on a synthetic polymer has been shown to act as an adjuvant to enhance the immune response, in addition to providing a controlled release. Aluminum salts can also be used as adjuvants to produce an immune response.
Optionally, one or more cytokines, such as interleukin (IL)-2, IL-6, IL-12, IL-15, RANTES, granulocyte macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, interferon (IFN)-α or IFN-γ, one or more growth factors, such as GM-CSF or G-CSF, one or more costimulatory molecules, such as ICAM-1, LFA-3, CD72, B7-1, B7-2, or other B7 related molecules; one or more molecules such as OX-40L or 41 BBL, or combinations of these molecules, can be used as biological adjuvants (see, for example, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90) with the disclosed immunogenic polypeptides. These molecules can be administered systemically (or locally) to the host. In several examples, IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1 B.7-2, OX-40L, 41 BBL, and/or ICAM-1 are administered.
Pharmaceutical compositions including the disclosed polypeptides, and/or nucleic acids encoding the polypeptides are also disclosed herein. The pharmaceutical compositions can include one or more of pharmaceutically acceptable carriers, adjuvants (such as those described above), a stabilizing detergent (such as polysorbate 80 (TWEEN® 80) (Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl); manufactured by ICI Americas, Wilmington, Del.), TWEEN® 40, TWEEN® 20, TWEEN® 60, ZWITTERGENT® 3-12, TEEPOL® HB7, and SPAN® 85 detergents, for example, in an amount of approximately 0.05 to 0.5%, such as at about 0.2%), a micelle-forming agent (such as PLURONIC® L62LF, L101, and L64 block copolymer, polyethylene glycol 1000, and TETRONIC® 1501, 150R1, 701, 901, 1301, and 130R1 block copolymer, for example, between 0.5 and 10%, or in an amount between 1.25 and 5%), and an oil (squalene, squalane, eicosane, tetratetracontane, glycerol, and peanut oil or other vegetable oils, for example, in an amount between 1 and 10%, or between 2.5 and 5%). In one embodiment, the pharmaceutical composition includes a mixture of stabilizing detergents, micelle-forming agent, and oil available under the name PROVAX® (IDEC Pharmaceuticals, San Diego, Calif.).
In some embodiments, a pharmaceutical composition includes one or more nucleic acids encoding a disclosed polypeptide. A therapeutically effective amount of the nucleic acid(s) can be administered to a subject in order to generate an immune response. In various embodiments, a nucleic acid encoding a biological adjuvant (such as those described above) can be cloned into same vector as a nucleic acid encoding a disclosed polypeptide, or the nucleic acid can be cloned into one or more separate vectors for co-administration. In addition, nonspecific immunomodulating factors such as Bacillus Calmette-Guerin (BCG) and levamisole can be co-administered.
One approach to administration of nucleic acids is direct immunization with plasmid DNA, such as with a mammalian expression plasmid. As described above, a nucleotide sequence encoding a disclosed polypeptide can be placed under the control of a promoter to increase expression of the molecule. Immunization by nucleic acid constructs is well known in the art and taught, for example, in U.S. Pat. No. 5,643,578 (which describes methods of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or a humoral response), and U.S. Pat. No. 5,593,972 and U.S. Pat. No. 5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to regulatory sequences enabling expression). U.S. Pat. No. 5,880,103 describes several methods of delivery of nucleic acids encoding immunogenic peptides or other antigens to an organism. The methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves), and immune-stimulating constructs, or ISCOMs, negatively charged cage-like structures of 30-40 nm in size formed spontaneously on mixing cholesterol and Quil A™ (saponin).
In another approach to using nucleic acids for immunization, a disclosed immunogenic polypeptide can also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirus or other viral vectors (such as those described above) can be used to express the peptide or protein. For example, vaccinia vectors and methods useful in immunization protocols are described in U.S. Pat. No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the peptides (see Stover, Nature 351:456-460, 1991).
In one embodiment, a nucleic acid encoding a disclosed immunogenic polypeptide is introduced directly into cells. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad's HELIOS™ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter. Typically, the DNA is injected into muscle, although it can also be injected directly into other sites
The amount of the disclosed immunogenic polypeptide, or nucleic acid molecule encoding the immunogenic polypeptide can vary depending upon the specific polypeptide(s), the route and protocol of administration, and the target population. In some embodiments, each dose includes about 1 μg to 1 mg of protein, such as from about 1 μg to about 500 μg, for example, from about 1 μg to about 100 μg, or about 1 μg to about 50 μg, such as about 1 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 40 μg, about 50 μg, about 75 μg, about 100 μg, about 200 μg, about 300 μg, about 400 μg, or about 500 μg. An optimal amount for a particular composition can be ascertained by standard studies involving observation of antibody titers and other responses in subjects (such as CTL or helper T cell responses).
The disclosed Env mosaic proteins (such as a set of three mosaic Env polypeptides) and/or nucleic acids encoding these proteins can be used in a multistep immunization regime. In some examples, the regime includes administering to a subject a therapeutically effective amount of a first immunogenic polypeptide (or mixture or set of immunogenic polypeptides) and boosting the immunogenic response with a second immunogenic polypeptide (or mixture or set of immunogenic polypeptides) after an appropriate period of time. This method of eliciting such an immune reaction is referred to as a “prime-boost” immunization regimen. Different dosages can be used in a series of sequential inoculations. Thus, a practitioner may administer a relatively large dose in a primary inoculation (prime) and then boost with relatively smaller doses. In some examples, the immunogenic polypeptide or mixture thereof administered in both the prime and boost inoculations are the same immunogenic polypeptide or mixture thereof. In other examples, the immunogenic polypeptide or mixture thereof administered in the boost is different from that administered in the prime inoculation.
The prime can be administered as a single dose or multiple doses, for example two doses, three doses, four doses, five doses, six doses, or more can be administered to a subject over days, weeks or months. The boost can be administered as a single dose or multiple doses, for example two to six doses or more can be administered to a subject over a day, a week or months. Multiple boosts can also be given, such one to five, or more. Different dosages can be used in a series of sequential inoculations. For example a relatively large dose in a primary inoculation and then a boost with relatively smaller doses. In some examples, there are weeks (for example, at least one week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 24 weeks, or more) between administration of a prime and a boost or between administration of two boosts in a prime-boost regimen The immune response against one or more of the Env mosaic proteins can be generated by one or more inoculations of a subject with an immunogenic composition disclosed herein.
The present disclosure is illustrated by the following non-limiting Examples.
This example describes the design strategy used to develop B cell-optimized mosaic Env proteins.
The B cell mosaic design was developed based on previous design strategies utilized for T cell mosaic polypeptides (Fischer et al., Nature Med. 13:100-106, 2007 and U.S. Pat. App. Publ. No. 2012/0231028, both of which are incorporated by reference herein in their entirety). The design strategy was modified as described below.
1. The capacity to incorporate structural information and alignments in the genetic algorithm for mosaic design (
2. A strategy for spanning hypervariable loop regions for the creation of an intact vaccine. HIV has four hypervariable regions within the variable loops V1, V2, V4, and V5 that typically evolve by insertion and deletion rather than just by base substitution. These short regions vary dramatically in length, charge, and number and location of N-linked glycosylation site motifs. They impact HIV neutralizing antibody recognition, and are essentially not alignable so could not be incorporated into part (1) above. Thus all hypervariable regions were excised from the alignments for the mosaic design phase, and if a position in the remaining core was close to these hypervariable regions, those positions were ignored during the mosaic design phase.
For the final designs, natural forms of these hypervariable regions that were short, relatively positively charged, with limited number of potential N-linked glycosylation sites (all desirable attributes in terms of antibody recognition based on neutralization data from large panels of antibodies compared to large panels of Envs tested in neutralization assays, provided by Dr. David Montefiori) were reintroduced. In addition, the frequencies of all peptide motifs of all lengths that were found within these regions were characterized, and natural variants with common motifs to span the hypervariable loops were favored.
3. A structural framework to define nearby regions in the protein. Ideally the HIV Env in its native form is needed, which requires a trimer structure. An early trimer model was used to enable the B cell mosaic design. 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 Tyr 712. The modified Env, designated Env(−)ΔCT, thus contained the complete ectodomain and transmembrane regions, and was purified from the plasma membrane of Env-expressing cells after solubilization in Cymal-5 detergent. This procedure ensured that the purified membrane-anchored Env(−)ΔCT trimers were glycosylated and passed the quality-control checkpoints of the secretory pathway (Moulard & Decroly (2000) Biochem. 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 detergent CYMAL®-5 was exchanged to CYMAL®-6 before preparation for cryo-EM imaging. The Env(-)ACT membrane glycoprotein, under the protection of CYMAL®-6, was flash-frozen on holey carbon film-coated EM grids and cryo-EM image data were collected at liquid nitrogen temperature. The imaging quality was found to be critically affected by the choice of the detergent and its concentration in the vitrified cryo-EM samples. A dataset of 90,306 single-particle images was assembled and subjected to multivariate data analysis, maximum-likelihood alignment and classification (Frank, J Three-dimensional electron microscopy of macromolecular assemblies: visualization of biological molecules in their native state. (Oxford Univ. Press, 2006); Sigworth (1998) J. Struct. Biol. 122:328-229; Scheres, et al. (2005) J. Mol. Biol. 348:139-149).
An initial model was generated by angular reconstitution from two-dimensional class averages 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). The model was then further refined by a projection-matching algorithm to a final resolution of 10.8 Å, measured by Fourier shell correlation (FSC) with a 0.5-cutoff criterion (Liao & Frank (2010) Structure 18:768-775). Using the 10.8-Angstrom model as a reference, by analyzing a large dataset of 582,914 individual Env trimer images (equivalent to 1,748,742 protomers), a cryo-EM map was obtained that was estimated to have a resolution of ˜6-Å based on the 0.5-cutoff of Fourier shell correlation. 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 Cα model to be traced manually in the program O (Jones, T. A. (2004) Acta Crystallogr. D 60:211-2125) (
This example describes the design of Env mosaic proteins.
Using the strategies described in Example 1, structure-based Env mosaic proteins were designed. The design was first optimized based on restricting the design to a single HIV clade, a regional approach for Southern African vaccine (C clade). A multi-clade global vaccine design was also developed, one that used only Transmitted/Founder virus sequences and one that used the full database. The Env structural mosaic protein sequences for use in the initial testing phase are disclosed herein as SEQ ID NOs: 1-8. These proteins were stably expressed and could be bound by critical broadly neutralizing antibodies, and sCD4. In particular, they were designed for use as sets of three proteins, and for many of the antibodies tested, they showed differential affinity for the antibody (see Example 3), a trait considered desirable and consistent with the design strategy, in that each mosaic displays different but common epitope variants. The design was based on the hypothesis that exposure to the common epitope variants in a vaccine will elicit antibodies with greater breadth, and the proteins disclosed herein were contemplated to be used in combinations of three proteins. Theoretical analysis suggests that the main advantage of the B cell mosaic design would be the possibility of a global vaccine, rather than a within-clade mosaic improving single-clade regional vaccine over a set of three natural C clade Envs (
A total of eight proteins were designed, which can be used in combinations of three antigens for use in single polyvalent vaccine. Particular combinations are described below.
1. Within clade C: Cmos3.1 (SEQ ID NO: 1), Cmos3.2 (SEQ ID NO: 2), Cmos3.3 (SEQ ID NO: 3). This set is a mosaic trio that was optimized to maximize the coverage of C clade transmitted viruses from the CHAVI/CAVD SGA sequence collections. They were made serially, first optimizing the coverage of Cmos3.1; then fixing it, and optimizing for the addition of Cmos3.2; then fixing both 3.1 and 3.2 and adding Cmos3.3. The serial addition did not compromise the total coverage relative to optimizing the three at once, and has the advantage of having the potential to be used as a single, double or tri-valent vaccine.
2. Three clade trimer: Cmos3.1 (SEQ ID NO: 1)+Bmos3.1 (SEQ ID NO: 4)+CRF01mos3.1 (SEQ ID NO: 5). This combination uses the optimal single mosaic from the transmitted virus sequences from three clades. The coverage of each of these clades is very good, and they were designed from transmitted viruses, which may be advantageous. The coverage of other clades (A, G, F, CRF02, and others) was not optimal, but better than inter-clade coverage of with natural C clade antigens or C clade mosaics (
3. Global mosaics: Mmos3.1 (SEQ ID NO: 6), Mmos3.2 (SEQ ID NO: 7), Mmos3.3 (SEQ ID NO: 8). There were not enough acute transmitted/founder sequences to give broad weight to some important epidemic lineages: clades A, G, F, and CRF01 and CRF02 are under-represented in the transmitted virus database, while B and C had a large enough sample size to work with. Thus chronic non-SGA sequences from the database were used to supplement the input data set for the under-represented lineages to create a global design. Thus this mosaic design set gives expanded coverage of all clades, but was not based on transmitted SGA sequences.
This example describes antigenicity of the global mosaic proteins described in Example 2.
The Mmos3.1, Mmos3.2, and Mmos3.3 polypeptides were expressed and tested in vitro using surface plasmon resonance (SPR). The dissociation constant of the polypeptides for various antibodies and epitopes was measured by SPR and is shown in Table 2. Low numbers represent antibodies with a slower off rates for the proteins. The antibodies names are listed on the left, the region of the HIV Env the target follows in parenthesis. CD4 is the receptor molecule on the HIV targeted T cell in natural infection. 17b is a monoclonal antibody that binds to the same region on the Env protein as the HIV-1 co-receptor molecule, and the capacity to bind to 17b and the co-receptor is CD4 inducible, hence the 17b (CD4i) nomenclature. The binding of CD4, and 17b after CD4 is bound, indicates that these proteins are well folded, and have native conformation in these regions, and the Env proteins undergo a biologically appropriate conformational change when CD4 is bound. The observation that these antibodies bind well to these proteins demonstrates that despite being artificial mosaic constructs, they form correctly folded proteins and retain the three-dimensional antigenic domains required for antibody binding.
Each of the three polypeptides was titrated on PG9, a very potent HIV-specific neutralizing antibody that typically has high dissociation constants (
Binding of Mmos3.1, Mmos3.2, and Mmos3.3 to A32, sCD4, and T8 was tested (
This example describes exemplary procedures for immunization of animals with the disclosed immunogenic polypeptides. Although particular methods are provided, one of ordinary skill in the art will appreciate that additional methods or variations of the described methods can also be utilized.
In some examples nucleic acid molecules encoding the disclosed immunogenic polypeptides are cloned into a plasmid or a viral vector (such as an adenoviral vector or a modified Ankara vaccinia virus vector). Study animals (for example, mice or monkeys) are administered plasmid or viral vector nucleic acid intramuscularly. Varying amounts of the nucleic acid can be administered, for example to test for an optimally effective amount.
In other examples nucleic acid molecules encoding the disclosed immunogenic polypeptides are cloned into an expression vector and expressed in a host cell. The polypeptides are purified using standard methods. Study animals (for example, mice or monkeys) are administered the polypeptides intramuscularly or subcutaneously. Varying doses are administered to determine optimal amounts for eliciting an immune response.
Immune responses elicited by the administered immunogenic polypeptides are assessed. For example, cellular immune responses are assessed using cytokine assays and/or interferon-γ ELISPOT assays. Humoral immune responses are assessed by direct ELISA utilizing one or more HIV proteins (such as a set of natural Env variants and/or the mosaic Env protein(s) administered to the animal). Neutralization assays (for example, a luciferase based pseudovirus neutralization assay) are also used to assess humoral immune responses.
This example describes exemplary methods for treating or inhibiting an HIV infection in a subject, such as a human subject, by administration of one or more of the immunogenic polypeptides or one or more nucleic acids encoding the immunogenic polypeptides disclosed herein. Although particular methods, dosages and modes of administrations are provided, one skilled in the art will appreciate that variations can be made without substantially affecting the treatment.
Briefly, the method includes screening subjects to determine if they have HIV, such as HIV-1 or HIV-2. Subjects having HIV are selected for further treatment. In one example, subjects are selected who have increased levels of HIV antibodies in their blood, as detected with an enzyme-linked immunosorbent assay, Western blot, immunofluorescence assay or nucleic acid testing, including viral RNA or proviral DNA amplification methods. In one example, half of the subjects follow the established protocol for treatment of HIV (such as a highly active antiretroviral therapy). The other half follow the established protocol for treatment of HIV (such as treatment with highly active antiretroviral compounds) in combination with administration of the agents including a therapeutically effective amount of a disclosed immunogenic polypeptide that induces an immune response to HIV. However, pre-screening is not required prior to administration of the compositions disclosed herein.
In particular examples, the subject is treated prior to diagnosis of AIDS with the administration of a therapeutically effective amount of one or more of the disclosed immunogenic polypeptides. In some examples, the subject is treated with an established protocol for treatment of AIDS (such as a highly active antiretroviral therapy) prior to treatment with the administration of a therapeutic agent that includes one or more of the disclosed immunogenic polypeptides. However, such pre-treatment is not always required and can be determined by a skilled clinician.
Following selection, an effective amount of one or more (such as three) immunogenic polypeptides disclosed herein, or one or more (such as three) nucleic acids encoding disclosed immunogenic polypeptides is administered to the subject (such as an adult human or a newborn infant either at risk for contracting HIV or known to be infected with HIV). Additional agents, such as anti-viral agents, can also be administered to the subject simultaneously or prior to or following administration of the disclosed agents. Administration can be achieved by any method known in the art, such as oral, inhalation, intravenous, intramuscular, intraperitoneal, or subcutaneous administration.
The amount of the immunogenic polypeptides (or nucleic acids encoding the polypeptides) administered to prevent, reduce, inhibit, and/or treat HIV or a condition associated with it depends on the subject being treated, the severity of the disorder and the manner of administration of the immunogenic composition. Ideally, an effective amount of the immunogenic composition is an amount sufficient to prevent, reduce, and/or inhibit, and/or treat the condition (for example, HIV) in a subject without causing a substantial cytotoxic effect in the subject. An effective amount can be readily determined by one skilled in the art, for example using routine trials establishing dose response curves. In addition, particular exemplary dosages are provided above. The therapeutic compositions can be administered in a single dose delivery, via continuous delivery over an extended time period, in a repeated administration protocol (for example, by a daily, weekly or monthly repeated administration protocol). In one example, a therapeutically effective amount of a disclosed antigen that induces an immune response to HIV is administered intravenously or intramuscularly to a human. As such, these compositions may be formulated with an inert diluent or with a pharmaceutically acceptable carrier. Immunogenic compositions can be taken long term (for example over a period of months or years).
Following the administration of one or more therapies, subjects having HIV (for example, HIV-1 or HIV-2) can be monitored for reductions in HIV levels, increases in a subjects CD4+ T cell count or reductions in one or more clinical symptoms associated with HIV infection. In particular examples, subjects are analyzed one or more times, starting 7 days following treatment. Subjects can be monitored using any method known in the art. For example, biological samples from the subject, including blood, can be obtained and alterations in HIV or CD4+ T cell levels evaluated.
In particular examples, if subjects are stable or have a minor, mixed or partial response to treatment, they can be re-treated after re-evaluation with the same or a different schedule and/or preparation of agents that they previously received for the desired amount of time, including the duration of a subject's lifetime. A partial response is a reduction, such as at least a 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 70% reduction of HIV viral load, HIV replication or combination thereof. A partial response may also be an increase in CD4+ T cell count such as at least 350 T cells per microliter.
This example describes methods that can be used to treat a subject that has or is at risk of having an infection from HIV that can be treated by eliciting an immune response, such as a neutralizing antibody response to HIV.
In particular examples, the method includes screening a subject having, thought to have or at risk of having a HIV infection. Subjects of an unknown infection status can be examined to determine if they have an infection, for example using serological tests, physical examination, enzyme-linked immunosorbent assay (ELISA), radiological screening, or other diagnostic techniques known to those of skill in the art. In some examples, subjects are screened to identify a HIV infection, with a serological test, or with a nucleic acid probe specific for a HIV nucleic acid. Subjects found to (or known to) have a HIV infection can be administered one or more disclosed immunogenic polypeptides (or nucleic acids encoding the polypeptides). Subjects may also be selected who are at risk of developing HIV for example, subjects exposed to HIV.
Subjects selected for treatment can be administered an effective amount of the disclosed immunogenic polypeptides or nucleic acids encoding the disclosed immunogenic polypeptides.
The particular dose can be determined by a skilled clinician. The polypeptides (or nucleic acids) can be administered in one or several doses, for example continuously, daily, weekly, or monthly. When administered sequentially the time separating the administration of the doses of the immunogenic polypeptides can be seconds, minutes, hours, days, or even weeks. Subjects are periodically tested for presence of HIV or HIV antibodies in their blood, as detected with an enzyme-linked immunosorbent assay, Western blot, immunofluorescence assay or nucleic acid testing, including viral RNA or proviral DNA amplification methods.
In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
This claims the benefit of U.S. Provisional Patent Application No. 61/884,696, filed Sep. 30, 2013, which is incorporated by reference herein in its entirety.
This invention was made with government support under Contract No. DE-AC52-06NA25396 awarded by the U.S. Department of Energy and grant number AI100645 from the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/058443 | 9/30/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61884696 | Sep 2013 | US |
