Patent Application
20080096187
The invention relates to the field of retroviruses, particularly lentivirus.
The antibody response to HIV-1 infection is typically vigorous and sustained but its effectiveness in virus containment in vivo is uncertain. We and others have shown in acutely infected individuals the rapid development of HIV-1 strain-specific neutralizing antibodies (Nab), and the equally rapid emergence of virus escape mutations (Albert et al. (1990) AIDS 4:107-112; Moog et al. (1997) J Virol 71:3734-3741; Wei et al. (2003) Nature 422:307-312; Richman et al. (2003) Proc Natl Acad Sci USA 100:4144-41492). Such strain-specific antibody responses are common, and they clearly drive virus selection in vivo (Wei et al. (2003) Nature 422:307-312; Richman et al. (2003) Proc Natl Acad Sci USA 100:4144-41492). More broadly reactive Nabs develop over longer periods (Pilgrim et al. (1997) J Infect Dis 176:924-932; Montefiori et al. (2001) J Virol 75:10200-10207; Parren et al (1999) Aids 13 Suppl A:S137-162). HIV-1 has evolved a variety of defense mechanisms to avoid antibody recognition, including epitope variation, oligomeric exclusion, conformational masking, glycan cloaking, and steric interference at the virus:cell interface (Kwong et al. (1998) Nature 393:648-659; Wyatt et al. (1998) Nature 393:705-711; Wyatt et al. (1998) Science 280:1884-1888; Kwong et al. (2002) Nature 420:678-682; Labrijn et al. (2003) J Virol 77:10557-10565; Burton et al. (2004) Nat Immunol 5:233-236; Zolla-Pazner et al (2004) Nat Rev Immunol 4:199-210), and together, they contribute to virus persistence in the face of an evolving antibody repertoire (Wei et al (2003) Nature 422:307-312; Richman et al. (2003) Proc Natl Acad Sci USA 100:4144-41492). But the precise nature of this evolving antibody response in vivo is incompletely understood. Analysis of HIV-1 specific monoclonal antibodies has revealed variable loop, CD4 binding site, chemokine co-receptor binding site, surface glycan, and membrane proximal gp41 domains as neutralization targets (reviewed in Burton et al. (2004) Nat Immunol 5:233-236; Zolla-Pazner et al (2004) Nat Rev Immunol 4:199-210), but the prevalence, titers, and breadth of polyclonal antibody responses to these epitopes in humans are generally unknown. This is in part a consequence of technical difficulty in identifying epitope-specific neutralizing antibody responses within a larger context of polyclonal neutralizing and non-neutralizing antibody reactivities (Broliden et al. (1992) Proc Natl Acad Sci USA 89:461-465; Scala et al. (1999) J Immunol 162:6155-6161; Opalka et al. (2004) J Immunol Methods 287:49-65).
It is clear that methods and compositions are needed to identify immunogenic, broadly-cross reactive epitopes on the HIV-1 envelope glycoprotein that might serve as targets of the adaptive humoral immune response in naturally-infected humans. Further needed are methods and compositions that allow for the detection of neutralizing HIV-1 antibodies.
Methods and compositions are provided to detect and identify HIV-1 binding antibodies. In specific methods and compositions, the HIV-1 binding antibody is a neutralizing antibody and/or a CD4-induced antibody. Such methods and compositions are capable of inducing a broadly protective response against HIV.
Methods are provided for detecting an HIV-1 binding antibody in a subject infected with human immunodeficiency virus-1 (HIV-1). The method comprises providing an envelope polypeptide or a functional variant thereof from a lentivirus that is not HIV-1, wherein the envelope polypeptide comprises at least one epitope recognized by an HIV-1 binding antibody. In specific methods, the envelope polypeptide is selected from the group consisting of an HIV-2 envelope polypeptide, a functional variant of the HIV-2 envelope, a Simian Immunodeficiency virus (SIV) envelope polypeptide or a functional variant of the SIV envelope polypeptide. The envelope polypeptide is contacted with an amount of bodily fluid from the subject. The HIV-1 binding antibody is detected. In specific methods, the method is capable of detecting the binding antibody present in the bodily fluid when present at a concentration of less than 0.1 μg/ml.
Methods are further provided for detecting CD4-induced antibodies in a subject infected with HIV-1. The method comprises providing an effective concentration of a soluble CD4/envelope complex. The complex comprises a soluble CD4 or a functional variant thereof and an envelope polypeptide from a lentivirus that is not HIV-1. The complex is contacted with an amount of bodily fluid from the subject; and, the CD4-induced antibodies are detected.
Methods for a diagnostic assay to monitor HIV disease in a subject or to monitor the response of a subject to immunization by an HIV vaccine are provided. The method comprises providing an envelope polypeptide or a functional variant thereof that is not from HIV-1 and comprises at least one epitope recognized by an HIV-1 binding antibody. The envelope polypeptide is contacted with an amount of bodily fluid from the subject, and the HIV-1 binding antibody in the bodily fluid of the subject is detected and HIV disease in the subject is thereby monitored or the response of the subject to immunization by an HIV vaccine is monitored. In specific methods, the envelope polypeptide is associated with a retrovirus.
Additional methods comprise providing an effective concentration of soluble CD4/envelope complex; contacting the complex with an amount of bodily fluid from the subject; and, detecting the CD4-induced antibodies in the bodily fluid of the subject and thereby monitoring HIV disease in the subject or the response of the subject to immunization by an HIV vaccine.
Additional methods include an assay for an HIV-1 binding antibody. The method comprises providing an envelope polypeptide or a functional variant thereof that is not from HIV-1 and the envelope polypeptide comprises an epitope recognized by an HIV-1 binding antibody. The envelope polypeptide is contacted with a composition comprising a candidate HIV-1 binding antibody; and, it is determined if the candidate antibody is an HIV-1 binding antibody.
Methods are also provided to determine an epitope for an HIV-1 binding antibody. The method comprises providing a population of envelope polypeptides or functional variants thereof that are not from HIV-1 and, wherein members of the envelope polypeptides in the population comprise at least one epitope recognized by the HIV-1 binding antibody and the envelope polypeptides in the population are substantially identical to one another. The population of envelope polypeptides is contacted with a composition comprising the HIV-1 binding antibody; and, the envelope polypeptide in the population that is recognized by the HIV-1 binding antibody is determined and the epitope for the HIV-1 binding antibody is thereby determined.
Methods are also provided to identify a soluble CD4 (sCD4) mimic. The method comprises providing an envelope polypeptide from a lentivirus that is not HIV-1; contacting the envelope polypeptide or a variant thereof with a candidate compound; and determining if the candidate compound interacts with the envelope polypeptide or functional variant thereof, wherein the interaction of the candidate compound and the envelope polypeptide or functional variant thereof increases the accessibility of an epitope or creates the epitope on the envelope polypeptide or the functional variant thereof, wherein the epitope is recognized by a CD4-induced antibody. In other methods, the CD4-induced antibody is from a subject infected with HIV-1, or the CD4-induced antibody was developed against an HIV-1.
A method to neutralize HIV-2 or SIV is also provided. The method comprises providing a composition comprising the HIV-2 or the SIV; providing to the composition an effective concentration of sCD4 or a functional variant thereof, wherein the sCD4 or the functional variant thereof is provided under conditions that allow for the interaction of the sCD4 or the functional variant thereof with the envelope polypeptide or the functional variant thereof of the HIV-2 or the SIV; and, providing to the composition an isolated CD4-induced antibody. In specific methods, the CD4-induced antibody is from a subject infected with HIV-1. In other methods, an effective concentration of the sCD4 is provided, and is some methods, the effective concentration of sCD4 comprises a concentration of about 1 nM to about 1000 nM.
Methods to alter the neutralization potential of a CD4-induced antibody elicited by HIV-1 are also provided. The method comprises providing an effective concentration of a soluble CD4/envelope complex; providing to the soluble CD4/envelope complex a CD4-induced antibody elicited by a HIV-1; and, thereby altering the neutralization potential of the CD4-induced antibody.
In specific methods, the envelope polypeptide employed in the methods is associated with a retrovirus. In other methods, the retrovirus is HIV-2 or SIV. In still other methods, the HIV-2 comprises the HIV-2 isolate 7312A or the ST isolate or a molecular clone thereof. In other methods, the retrovirus comprises a pseudotyped primate lentivirus. In other methods, the envelope polypeptide comprises an amino acid sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 2, 3, 4, or 5.
In yet other methods, the epitope recognized by the HIV-1 binding antibody is found within gp41, gp120 or the membrane proximal external region of gp41. In still further methods, the epitope recognized by the HIV-1 binding antibody comprises a 4E10 epitope, a 2F5 epitope, or a Z13 epitope. The epitope recognized by the HIV-1 binding antibody can be homologous or heterologous to the envelope polypeptide.
Compositions of the invention include a chimeric polynucleotide comprising a nucleotide sequence encoding an envelope polypeptide or functional variant thereof that is not from HIV-1, wherein the amino acid sequence further comprises a heterologous epitope recognized by an HIV-1 neutralization antibody.
Additional compositions include a chimeric polypeptide comprising an amino acid sequence of an envelope polypeptide or a functional variant thereof that is not from HIV-1, wherein the amino acid sequence further comprises a heterologous epitope recognized by an HIV-1 neutralization antibody.
Cells, viruses, kits, and directs for their use comprising the various compositions of the invention are further provided. Additional compositions include a kit comprising a soluble CD4/envelope complex and directions for use.
The present inventions now will be described more fully hereinafter with reference to the accompanying examples, in which some, but not all claims of the invention are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more than one element.
With many HIV-1 vaccine candidates currently in the research pipeline, methods are needed for detecting and quantifying epitope-specific neutralizing antibody responses in naturally-infected individuals and vaccinated subjects. HIV-1 and HIV-2 share less than 50% sequence similarity in envelope and they generally exhibit little cross-neutralization. The present invention demonstrates the successful identification of HIV-1 neutralization epitopes in, or molecularly engineered into, functional envelope glycoproteins from non-HIV-1 envelope polypeptides. Accordingly, various methods and compositions are provided for the detection and/or characterization of an HIV-1 binding antibody, particularly HIV-1 neutralizing antibodies.
As used herein an “HIV-1 binding antibody” comprises an antibody that specifically interacts with an epitope of HIV-1. In specific embodiments, the HIV-1 binding antibody interacts with an epitope of the envelope polypeptide of HIV-1. An HIV-1 binding antibody that can neutralize a virus is referred to herein as an “HIV-1 neutralizing antibody.” Additional HIV-1 binding antibodies include CD4-induced antibodies, and in more specific embodiments, the CD4-induced antibodies are neutralizing antibodies.
By “specifically interacts” is intended that the antibody that recognizes the epitope of an HIV-1 envelope polypeptide forms a specific antibody-antigen complex with that epitope (either in an in vitro or in vivo setting) when the epitope is contained in an envelope polypeptide that is not from HIV-1. Thus, the HIV-1 binding antibody binds preferentially to the non-HIV-1 envelope polypeptide comprising the HIV-1 epitope. By “binds preferentially” is meant that the antibody immunoreacts with (binds) substantially more of the non-HIV-1 envelope polypeptide comprising the HIV-1 epitope than the non-HIV-1 envelope polypeptide lacking the epitope, when both polypeptides are present in an immunoreaction admixture. Substantially more typically indicates at least greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater of the immunoprecipitated material is the non-HIV-1 envelope polypeptide comprising the HIV-1 epitope.
Methods are provided for the detection of an HIV-1 binding antibody (i.e., a neutralizing antibody) in a subject infected with HIV-1. The method comprises providing an envelope polypeptide or a functional variant thereof from a lentivirus that is not HIV-1, where the envelope polypeptide comprises at least one epitope recognized by an HIV-1 binding antibody. The envelope polypeptide is contacted with an amount of bodily fluid from the subject, and the HIV-binding antibodies are detected. Methods for contacting the envelope polypeptide with the HIV-1 binding antibody include in-vitro binding studies such as those discussed in Xiang et al. (2002) AIDS Res Hum Retroviruses 18:1207-17; Cavacini et al. (2003) AIDS 17:1863; and Xiang et al. (2003) Virology 315:124-34, each of which is herein incorporated by reference. Alternatively, the envelope polypeptide can be in association with a lipid bilayer in a number of different ways, so long as the envelope polypeptide exists in one or more confirmation that is similar to the envelope protein in its native environment. In one method, the envelope polypeptide is associated with a retrovirus. By “associated” is intended the envelope polypeptide is present on the surface of the retrovirus. In this method, a composition comprising a retrovirus having an envelope polypeptide from a primate lentivirus that is not HIV-1 is provided. An amount of bodily fluid from the subject is contacted with the envelope polypeptide, and the HIV-1 binding antibodies are detected. Any bodily fluid can be employed in the methods of the invention, including, but not limited to, serum, plasma, semen, milk, etc. If the HIV-1 binding antibodies are present in the patient bodily fluid, the antibodies will interact with the epitope. In specific embodiments, the interaction of the antibody with the epitope results in the neutralization of the virus in the sample.
Methods to assay for an interaction of an HIV-1 binding antibody with an epitope on the envelope polypeptide are known. For example, formation of an antibody-antigen complex using a number of well-defined diagnostic assays can be used including conventional immunoassay formats to detect and/or quantitate antigen-specific antibodies. Such assays include, for example, enzyme immunoassays, e.g., ELISA, cell-based assays, flow cytometry, radioimmunoassays, and immunohistochemical staining. Numerous competitive and non-competitive protein binding assays are known in the art and many are commercially available. Representative assays include, for example, various binding assays with chemokine receptors (CCR5 or CXCR4), gp41, characterized domains of these polypeptides, and competitive binding assays with characterized HIV-1 binding antibodies. In addition, if the envelope polypeptide is associated with a retrovirus, “neutralization” of the virus and thereby reducing the establishment of HIV infection and/or reducing subsequent HIV disease progression (i.e., reduces the severity of the symptoms of the HIV infection) in a sample when compared to a control virus lacking the HIV-1 binding antibody can also be assayed. A reduction in the establishment of HIV infection and/or a reduction in subsequent HIV disease progression encompasses any statistically significant reduction in HIV activity in the sample. Such HIV-1 binding antibodies that neutralize the virus are referred to herein as “HIV-1 neutralizing antibodies.” Methods to assay for the neutralization activity include, but are not limited to, a single-cycle infection assay as described in Martin et al. (2003) Nature Biotechnology 21:71-76. In this assay, the level of viral activity is measured via a selectable marker whose activity is reflective of the amount of viable virus in the sample, and the IC50 is determined. In other assays, acute infection can be monitored in the PM1 cell line or in primary cells (normal PBMC). In this assay, the level of viral activity can be monitored by determining the p24 concentrations using ELISA. See, for example, Martin et al. (2003) Nature Biotechnology 21:71-76, herein incorporated by reference. Further methods include those employing the adherent HeLa cell-derived JC53BL-13 cell line (NIH AIDS Research and Reference Reagent Program Catalogue No. 8129, TZM-bl) as described in Wei et al. (2003) Nature 422:307-312, herein incorporated by reference.
The method of detecting the HIV-1 binding antibodies is very sensitive and is capable of detecting HIV-1 binding antibody concentrations of less than about 1 μg/ml, less about 0.5 μg/ml, less than about 0.3 μg/ml, less than about 0.2 μg/ml, less than about 0.1 μg/ml, less than about 0.09 μg/ml, less than about 0.08 μg/ml less than about, 0.07 μg/ml, less than about 0.06 μg/ml, less than about 0.05 μg/ml, less than about 0.04 μg/ml, less than about 0.03 μg/ml, less than about 0.02 μg/ml, less than about 0.01 μg/ml, less than about 0.009 μg/ml, less than about 0.005 μg/ml, or less than about 0.001 μg/ml or less.
In other methods the HIV-1 binding antibody is a CD4-induced antibody. In specific embodiments, the CD4-induced antibody is a neutralizing antibody. Accordingly, methods are also provided for the detection of CD4-induced antibodies in a subject infected with HIV-1. The method comprises providing an effective concentration of a soluble CD4/envelope complex. The complex comprises a soluble CD4 or a functional variant thereof and an envelope polypeptide from a lentivirus that is not HIV-1 or a functional variant thereof. The soluble CD4/envelope complex is contacted with an amount of bodily fluid from the subject and the CD4-induced antibodies are detected.
As used herein, a “soluble CD4/envelope complex” comprises a soluble CD4 or a functional variant thereof and an envelope polypeptide from a primate lentivirus that is not HIV-1 (i.e., HIV-2, SIV, SRV-1, SUV-2, Simian human immunodeficiency virus, and HIV-3) or a functional variant thereof. The components of the complex can interact through covalent or non-covalent interactions. In specific embodiments, the interactions between the sCD4 and the envelope polypeptides are non-covalent. Methods for forming such a complex include those discussed in Xiang et al. (2002) AIDS Res Hum Retroviruses 18:1207-17; Cavacini et al. (2003) AIDS 17:1863; and Xiang et al. (2003) Virology 315:124-34, each of which is herein incorporated by reference.
As used herein, the term “CD4-induced antibody” comprises an antibody that interacts with an epitope of the envelope polypeptide of a primate lentivirus, where the epitope is created or exposed or the accessibility of the epitope is increased in the presence of an effective concentration of soluble CD4 or a functional variant of soluble CD4. The created epitope or the epitope having the increased accessibility under these conditions is referred to herein as a “CD4-induced epitope.” Methods to measure the creation of an epitope or an increase in exposure or accessibility of an epitope are discussed elsewhere herein. Briefly, binding assays with compounds that interact with the exposed epitope can be preformed. Such compounds include, for example, characterized CD4-induced antibodies and chemokine receptors. In the method described above, soluble CD4 interacts with the envelope polypeptide and increases the accessibility of a CD4-induced epitope. If CD4-induced antibodies are present in the patient bodily fluid, the antibody will interact with the epitope. In specific embodiments, the interaction of the antibody with the epitope results in the neutralization of the virus in the sample. It is recognized that specific methods of the invention can be performed in-vitro or in-vivo.
Methods to assay for an interaction of a CD4-induced antibody with an epitope on the envelope polypeptide include, for example, various binding assays with chemokine receptors (CCR5 or CXCR4) or with characterized CD4 induced antibodies. In addition, if the envelope polypeptide is associated with a retrovirus, “neutralization” of the virus can be assays. Such methods are discussed in detail elsewhere herein.
In specific methods of the invention, the HIV-1 binding antibody, neutralizing antibody, and/or CD4-induced antibody is isolated. An “isolated” antibody is substantially or essentially free from components that normally accompany or interact with the antibody as found in its naturally occurring environment. Thus, an isolated or purified antibody is substantially free of other cellular material or culture medium. An antibody that is substantially free of cellular material or culture medium includes preparations of antibody having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.
The envelope polypeptide employed in the methods may be in the either in the glycosylated or deglycosylated form. In addition, the envelope of the invention can be an envelope polypeptide from any lentivirus or any primate lentivirus. In specific methods, the envelope polypeptide is from any primate lentivirus that is not HIV-1. Such primate lentivirus include, for example, HIV-2 (Isolate BEN), HIV-2 (Isolate CAM2), HIV-2 (Isolate D194), HIV-2 (Isolate D205,7), HIV-2 (Isolate GHANA-1), HIV-2 (Isolate ROD); Simian AIDS retrovirus (SRV-1) such as, SIV (AGM155), SIV (AGM266 isolate), SIV (AGM3 isolate), SIV (AGM385 isolate), SIV (F236/SMH4 isolate, Sooty Mangabey), SIV (TyO-1 isolate) and SIVagm; Simian immunodeficiency virus, such as, SIV (1A11 isolate), SIV (isolate African mandril), SIV (AGM/clone Gri-1), SIV (vervet), SIV (Tantalus), SIV, STM isolate, SIV, 17E-Cl, SIV Qu, SIVdeb, SIVmac, SIVMND, SIVmon, SIVsm; Simian immunodeficiency virus 2; and Simian-Human immunodeficiency virus.
In specific methods, the envelope polypeptide is from HIV-2. For example, in one method, an HIV-2 envelope polypeptide or functional variants thereof is used. By “HIV-2 envelope polypeptide” or “envelope encoded by an HIV-2 polynucleotide” is intended the form of the HIV-2 envelope polypeptide or polynucleotide encoding the same in the HIV-2 viral isolate 7312A. The amino acid of the envelope polypeptide of the HIV-2 isolate 7312A is set forth in
Variants of the HIV-2 envelope polypeptide are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, they continue to interact with CD4 and/or facilitate virus fusion and/or facilitate viral entry into a permissive cell. It is further recognized that the viral envelope polypeptide is produced as a precursor (gp160) that is subsequently cleaved into two parts, gp120 which binds CD4 and chemokine receptors, and gp41, which is anchored in the viral membrane and mediates viral fusion. Variants of the HIV-2 envelope polypeptide encompass fragments of HIV-2 envelope including, for example, gp41, gp120 or any other fragment that retains the necessary activity. The amino acid sequence comprising gp41 and gp120 is denoted in
Variants of HIV-2 envelope polypeptide are known. See, for example,
In another method an SIV envelope polypeptide or functional variants thereof is used. By “SIVsm envelope polypeptide” or “envelope encoded by an SIVsm envelope polynucleotide” is intended the form of the SIVsm envelope polypeptide or polynucleotide encoding the same in SIVsm PBJ1.9. The amino acid of the envelope polypeptide of the SIVsm PBJ1.9 is set forth in SEQ ID NO:3 and the nucleotide sequence encoding this polypeptide is set forth in SEQ ID NO:22. In other methods, a SIVsm envelope polypeptide, polynucleotide, or a functional variant thereof. See, also, Israel et al. (1993) AIDS Res. Hum. Retroviruses 9:277-286; Hirsch et al. (1998) Nat. Med. 4(12):1401-8; Mahalingam et al. (2001) J. Virol. 75(1):362-74, each of which is herein incorporated by reference.
By “SIVagm envelope polypeptide” or “envelope encoded by an SIVagm polynucleotide” is intended the form of the SIVagm envelope polypeptide or polynucleotide encoding the same in SIVagmVer155. The amino acid sequence of the envelope polypeptide of SIVagmVer155 is set forth in SEQ ID NO:4. See, also, Johnson et al. (1990) J. Virol. 64 (3), 1086-1092, herein incorporated by reference. Other envelope polypeptides from SIVagm are known. For example, the amino acid sequence for the envelope polypeptide from SIVagmTAN is provided in SEQ ID NO:5. See, also, Soares et al. (1997) Virology 228 (2): 394-399.
Variants of the SIV envelope polypeptide are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, they continue to interact with CD4 and/or facilitate virus fusion and/or facilitate viral entry into a permissive cell. Variants of the SIV envelope polypeptides encompass fragments of SIV envelope including, for example, gp41, gp120 or any other fragment that retains the necessary activity. The amino acid sequence of gp41 and gp120 are denoted in
In still other methods, the envelope polypeptide is associated with a retrovirus. Any retrovirus can be used including lentiviruses and primate lentiviruses. The term HIV refers to all strains, isolates, and molecular clones of human immunodeficiency virus. Many different retroviruses can be used in the methods of the invention. For example, in one method, the retrovirus having the HIV-2 envelope polypeptide comprises an HIV-2 virus, including any primary HIV-2 isolates, laboratory strains, or molecular clones derived there from. In addition, the HIV-2 can be infectious or non-infectious. HIV-2 viruses include, but are not limited to, UC1, HIV-2 MS, CBL 20. In another method, the HIV-2 virus employed is HIV-2 7312A one of its molecular clones including, for example, pJK7312A or V434M. V434M has a single amino acid change from V→M at amino acid 434 in the envelope polypeptide. The clone has particular sensitivity in the detection of CD4 induced antibodies. In still other methods, the HIV-2 virus is HIV-ST or its molecular clone pJSP4-27(ST/SXB1). See, the Experimental section for a complete description of these particular molecular clones. See, also Gao et al. Nature (1992) 358:495-499 and found in GenBank Accession No. L36874 and in the Los Alamos HIV database operated by the University of California at “.hiv.land.gov/content/index”, herein incorporated by reference. Similarly, a retrovirus having the SIV or SRV-1 envelope polypeptide can comprise an SIV or an SRV-1 virus, including any primary SIV or SRV-1 isolates, laboratory strains, or molecular clones. In addition, the SIV or SRV-1 can be infectious or non-infectious.
In still other methods, the retrovirus having the envelope polypeptide or the functional variant thereof comprises a retrovirus that has been pseudotyped with the envelope polypeptide from the primate lentivirus that is not HIV-1 or functional variant thereof. Retrovirus that can be used in these methods include, but are not limited to, lentiviruses, such as, bovine lentivirus, equine lentivirus, feline lentivirus, ovine/caprine lentivirus, and primate lentivirus. Primate lentivirus that can be used include, HIV-1, HIV-2, HIV-3, SRV-1, SIV, SIV-2 and simian-Human immunodeficiency virus. In specific methods, the SIVsm and SIVagm are used.
In addition, the retrovirus employed in the methods may be infectious or non-infectious. For example, non-infectious HIV-1 strains include 8E5/LAV virus (Folks et al. (1986) J. Exp. Med. 164:280-290; Lightfoot et al. (1986) J. Virol. 60:771-775 and Gendelman et al. (1987) Virology 160:323-329), and HIV-1 JR-FL. In still other methods, the virus pseudotyped with the envelope polypeptide from the primate lentivirus or the functional variant thereof is an infectious laboratory-adapted or a primary isolate of HIV-1, HIV-2, SIV, or SRV-1. See, for example, Haddrick et al. (1996) J. Virol. Methods 61:89-93 and Yamshchikov et al. (1995) Virology 21:50-58. It is further recognized that sequences from many strains of retroviruses are publicly available on Genbalik and primary field isolates of HIV are available from the National Institute of Allergy and Infectious Diseases (NIAID). Such strains are also available from the World Health Organization (WHO) [Network for HIV Isolation and Characterization, Vaccine Development Unit, Office of Research, Global Programme on AIDS, CH-1211 Geneva 27, Switzerland]. Methods of pseudotyping viruses are known in the art. See, for example, US Application No. 20040033604, U.S. Application No. 200330203489, Schauber et al. (2004) Gene Ther 11:266-75, and Kafri et al. (2004) Methods Mol. Biol. 246:376-90.
The envelope polypeptide employed in specific methods of the invention comprises at least one epitope that is recognized by an HIV-1 binding antibody. Various methods to determine if such an epitope is present in the envelope polypeptide are discussed in detail elsewhere herein. It is recognized that the epitope recognized by the HIV-1 binding antibody can be homologous or heterologous to the envelope polypeptide that it is contained in. A homologous epitope for an HIV-1 binding antibody is present in the native envelope polypeptide. A heterologous epitope for an HIV-1 binding antibody is not present or found in an alternative location in the native envelope polypeptide. Polypeptides comprising such heterologous epitopes are referred to herein as “chimeric polypeptides.”
A variety of epitopes for HIV-1 binding antibodies are known in the art. Such epitopes are found both in gp160, gp120, gp41. See, for example, HIV Molecular Immunology (2002) Korber et al. ed., Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N. Mex. LA-UR 03-5816, which is herein incorporated by reference in its entirety. In specific embodiments, the epitope of the HIV-1 binding antibody is in gp41. For example, the epitope in the N-terminal hydrophobic fusion peptide of gp41 (about amino acids 512 to about 527 of SEQ ID NO:10), the disulfide-loop region of gp41 that links the N-HR and C-HR regions (about amino acids 581 to about 628 of SEQ ID NO:10), the N-HR region of gp41 (about amino acids 546 to about 581 of SEQ ID:10), the C-HR of gp41 (about amino acids 628 to about 661 of SEQ ID NO: 10), the membrane proximal region of gp41 (about amino acids 657 to about amino acids 684 of SEQ ID NO:10).
As used herein, an “MPER region” comprises the MPER region found in HIV-1 YU-2 (i.e., N-LALDKWASLWNWFDITKWLWYIK-C (SEQ ID NO:38)). A functional variant of an MPER region will continue to be recognized by an HIV-1 binding antibody. Method to assay for the binding of the HIV-1 binding antibody are discussed elsewhere herein as are methods to determine if the variant sequence is immunologically equivalent. Such variants can include internal and/or terminal additions, deletions, and/or substitutions. The variants can differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acids. Variants of the MPER region are know. See, for example,
Functional variants of the MPER region comprise substitutions, additions, and/or deletions (including internal or terminal alterations or both).
Epitopes within the membrane proximal region of gp41 can be found, for example, between about amino acids 657 to 675, about amino acid 670 to 684, about amino acids 665 to about 680, or about amino acids 667 to about 681 of SEQ ID NO:10. See, Follis et al. (2002) J. of Virology 76:7356-7362 for additional domains of gp41 that are of interest. In other embodiments, epitope of the HIV-1 binding antibody is found in the bridging sheet, variable loop 1, variable loop 2, variable loop 3, variable loop 4, the chemokine receptor binding site, or the CD4 binding site. See, for example,
While any epitope for an HIV-1 binding antibody may be used, of particular interest is a neutralizing epitope found in the HIV-1 envelope polypeptide. Epitopes of interest include, but are not limited to, the 4E10 epitope (SEQ ID NO:15), the Z13 epitope (SEQ ID NO:15) and the 2F5 epitope (SEQ ID NO:16). See, for example, U.S. Publication No. 20030157063, Muster et al (1993) J. Virol. 67:6642-6647, Zwick et al. (2001) J. Virology 75:10892-10905, Ferrantelli et al. (2002) Curr. Opin. Immunol. 14:495-502, and Wang et al. (2003) Curr. Phamm. Des. 9:1771-87. Each of these epitopes is denoted in
It is further recognized that immunological equivalent epitopes for the HIV-1 binding antibodies discussed above are known and can be used in the methods and compositions of the invention. Immunologically equivalent epitopes for 2F5 are known. See, for example, U.S. Application Publication No. 20030157063, Kattinger et al. (1992) Septime Colloque des Cent Gardes, 299-303, EP-0570357, and Zwick et al. (2001) J. Virology 75:10892-10900 which disclose immunologically equivalent epitopes of the 2F5 epitope. Such immunologically equivalent epitopes, while differing in their amino acid sequence continue to be recognized by the 2F5 monoclonal antibody (Virus Testing Systems, Houston, Tex., USA). Immunologically equivalent epitopes for 4E10 and Z13 are also known. See, for example, Zwick et al. (2001) J. Virology 75:10892-10900. Again, such immunologically equivalent epitopes, while differing in their amino acid sequence continue to be recognized by the 4E10 monoclonal antibody or the Z13 antibody. Accordingly, immunologically equivalent epitopes can differ from the epitope set forth in SEQ ID NO: 15 and 16 by at least 1, 2, 3, 4, 5, 6, 7, 8 or more amino acids. The differences can be generated by amino acid substitutions, deletions and insertions. Method to determine if two epitopes are immunologically equivalent are known in the art. See, for example, U.S. Application Publication No. 20030157063, EP-0570357 and Zwick et al. (2001) J. Virology 75:10892-10900, all of which are herein incorporated by reference.
Many HIV-1 binding antibodies are known in the art and can be employed in the methods and compositions of the invention. The term “antibody” as used herein includes monoclonal, polyclonal, chimeric, single chain, bispecific, simianized, and humanized or primatized antibodies as well as Fab fragments, such as those fragments which maintain the binding specificity of the antibodies to the epitope of the HIV-1 envelope polypeptide. Accordingly, the invention contemplates the use of single chains such as the variable heavy and light chains of the antibodies. Generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art. Various CD4-induced antibodies are known in the art and can be employed in the methods of the invention, including, but not limited to 17b (Zhang et al. (1999) Biochemistry 38:9405-16), 21c, 19e, E51 (Xiang et al. (2003) Virology 315:124), X5 (Darbha et al. (2004) Biochemistry 43:1410), ED49, and ED47.
In the methods of the invention, the envelope polypeptide or the functional variant thereof is contacted with compositions that may comprise the HIV-1 binding antibody. It is recognized that such methods of the invention will be carried out in an appropriate buffer and at the appropriate temperature to promote the desired interaction and to allow the necessary activities to be measured. One of skill will be capable of determining the appropriate buffers and temperatures that will promote the desired interaction. See, for example, Moore et al. (1990) AIDS 4:297-303 and Dey et al. (2003) Journal of Virology 77:2859-2865. In one embodiment, the detection of HIV-1 binding antibodies is performed under the conditions outlined in Wei et al. (2003) Nature 422:307-312, herein incorporated by reference.
As discussed above, in one method of the invention, HIV-1 binding antibodies (i.e., binding antibodies, neutralizing antibodies, and/or CD4-induced antibodies) in a subject infected with HIV-1 are detected. In other methods, the titer of the HIV-1 binding antibody in a sample is determined. In still other methods, the HIV-1 binding antibody is isolated and characterized. The subject can be any mammal infected with HIV-1 including humans and non-humans, such as, monkeys. Several methods can be used to detect the presence of the HIV-1 binding antibodies. For example, detection of the antibodies can be determined by assaying for a decrease in infectivity of the retrovirus (i.e., the neutralization of the retrovirus). Any statistically significant decrease when compared to the appropriate control indicates that HIV-1 neutralizing antibodies are present in the bodily fluid of said patient. Methods to determine the infectivity of the retrovirus having the envelope polypeptide have been discussed in detail elsewhere herein. Other methods to detect the HIV-1 binding antibodies include competitive binding assays with the chemokine receptors (i.e., CCR5 and CXCR4) or with characterized HIV-1 binding antibodies, or the use of cell fusion assays. Each of these assays is described in detail, for example, in Martin et al. (2003) Nature Biotechnology 21:71-77.
As discussed above, methods are provided for the detection of CD4-induced antibodies, which employs the use of an effective concentration of a soluble CD4/envelope complex. CD4 is a member of the immunological superfamily and it comprises an extracellular region comprising four immunoglobulin-like domains (D1-D4), a membrane spanning region, and a charged cytoplasmic domain. The cDNA encoding CD4 is found in Maddon et al. (1985) Cell 42:93 and in Genbank Accession No. RWHUT4, both of which are herein incorporated by reference. The full length CD4 is set forth in SEQ ID NO:6. In human CD4, amino acid residues from about 30 to about 60 play a role in the interaction of CD4 with HIV-1 gp120. Residue Phe-43 of hCD4 is believed to play a role in the CD4/gp120 interaction. See, for example, Clayton et al. (1988) Nature 22:363-6, Jameson et al (1998) Science 240:1335-1339, Piatier-Toneua et al. (1991) PNAS 88:6858-6862.
As used herein, “soluble CD4” or “sCD4” refers to the human form of CD4 that comprises a CD4 polypeptide that lacks a portion of the hydrophobic anchor domain such that the soluble CD4 or biologically active variants thereof are soluble in water-based pharmaceutical preparations (or pharmaceutically acceptable solvents or compositions which include components in addition to water) and in physiological fluids, including plasma, at a level which is sufficient to achieve an effective concentration. As used herein, by “sCD4” is intended the form of sCD4 set forth in SEQ ID NO:1.
Variants of the soluble CD4 polypeptide are biologically active, that is they continue to possess the desired biological activity of the native sCD4 protein, that is, they continue to interact with the envelope polypeptide and/or a functional variant thereof, wherein the interaction of the sCD4 variant with the envelope polypeptide or the functional variant thereof exposes or increases the accessibility of a sCD4-inducible epitope on the envelope polypeptide or the functional variant thereof. Variants of sCD4 proteins include those in which part or the entire transmembrane domain of the primary structure of CD4 has been deleted, for example through truncation of the coding sequence. The cytoplasmic domain of the protein may likewise be deleted without the loss of the desired biological activity of HIV envelope binding.
CD4 and recombinant CD4 that is synthesized in recombinant eukaryotic cells is a glycoprotein. It is recognized that the native full-length CD4, the sCD4, or the functional variant thereof can be glycosylated. See, Maddon et al. (1985) Cell 42:93 and U.S. Pat. No. 5,234,905. It is further recognized that the exact oligosaccharide structure of the glycoprotein may vary with respect to sugars present, the glycosylation enzymes present and the relative proportions of each according to the choice of the particular eukaryotic cell in which the recombinant CD4 (or soluble CD4) is synthesized. Soluble CD4 molecules capable of being glycosylated when synthesized in appropriate host cells are described in Smith et al. (1987) Science 238:1704; Fisher et al. (1988) Nature 331:76; Hussey et al. (1988) Nature 331:78; EP Publication No. 385 909; Deen et al. (1988) Nature 331:82-84; all of which are incorporated by reference herein.
Functional variants of soluble CD4 include, for example, conservative amino acid alterations to the polypeptide of SEQ ID NO:1 along with functional variants that interact with the external envelope glycoprotein, gp120, derived from HIV. Additional functional variants of sCD4 include various peptide variants such as CD4M9 (a 28 amino acid peptide) and CD4M33. See, for example, Martin et al. (2003) Nature Biotechnology 21:71-76, herein incorporated by reference. In addition, a tetrameric form of sCD4 (Allway et al. (1995) AIDS Res. Hum. Retroviruses 69:6609-6617) and a dodecameric form of sCD4 (Arthos et al. (2002) J. Biol. Chem. 277:11456-11464) can also be employed. Other functional variants are disclosed in WO-97/08574, Chao et al. (1989) J. Biol. Chem. 264:5812, Peterson and Seed (1988) Cell 54:65-72, U.S. Pat. No. 5,767,022, and U.S. Pat. No. 5,234,905, all of which are herein incorporated by reference.
As used herein, an “effective concentration” of a sCD4/envelope complex or of a soluble CD4 or a functional variant thereof comprises a concentration sufficient to create, expose and/or increase the accessibility of an epitope recognized by a soluble CD4-induced antibody. An effective concentration of soluble CD4 or an active variant thereof include final soluble CD4 concentrations of about 0.1 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 140 nM, 160 nM, 180 nM, 200 nM, 220 nM, 220 nM, 260 nM, 280 nm, 300 nM, 350 nM, 400 nM, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1200 nm, 1500 nm, 1800 nm, 2000 nm, 2500 nm, 4000 nm or greater. In other embodiments, the effective concentration of soluble CD4, or the functional variants or mimic thereof include final concentrations between about 0.1 nM and about 1 mM, between about 1 nM and 5000 nM, between about 1 nM and 4000 nM, between about 1 nM and 2000 nM, between about 1 nm and 10001 nM, between about 280 nM and 450 nm, and between about 1 nm and 100 nm. One of skill will recognize that depending on the sCD4 or functional variant thereof and the specific assay employed, the effective concentration of may vary.
Methods to determine if an effective concentration of soluble CD4 has been provided include, but are not limited to, performing a neutralization assay in which the target virus is incubated in the presence of soluble CD4 or a functional variant thereof. The mixture is exposed to a CD4-induced antibody. The infectivity of the target virus is determined in the presence and absence of the soluble CD4 or the functional variant thereof. An effective concentration of soluble CD4 or its functional variant will be sufficient to neutralize the virus. Methods to assay for viral neutralization are discussed elsewhere herein. Alternatively, methods to determine if an effective concentration of soluble CD4 or an effective concentration of a sCD4/envelope complex has been provided also includes various binding assays, for example, with the chemokine receptors or with a characterized CD4-induced antibody. Such methods are discussed elsewhere herein.
When CD4-induced antibodies are to be detected, the sCD4 or the functional variant thereof is provided under conditions that allow for the interaction of the sCD4 or the functional variant thereof with the envelope polypeptide or the functional variant thereof from the non-HIV-1 primate lentivirus. Thus, methods of the invention will be carried out in an appropriate buffer and at the appropriate temperature to promote the desired interaction and to allow the necessary activities to be measured. In the methods disclosed herein, the order in which the sCD4 or variant thereof and the sample containing the CD4-induced antibodies are provided in the methods disclosed herein can be varied. For example, in some methods, the sCD4/envelope complex is formed prior to the addition of a sample bodily fluid sample or a sample having the CD4-induced antibody. In specific methods, the sCD4 is incubated with the envelope polypeptide to form the sCD4/envelope complex for any period of time sufficient to allow for the desired interaction including, for example, 0.1 hr, 0.5 hr, 1 hr, 1.5 hr or greater. In other methods, the sample having the CD4-induced antibody is contacted with the envelope polypeptide prior to the addition of the sCD4 or the variant thereof. In yet other methods, the addition of sCD4, the envelope polypeptide, and the CD4-induced antibodies occurs simultaneously.
In still further methods, soluble CD4 is not required to expose, create or increase the accessibility of the epitope that is recognized by the CD4-induced antibody. In this method, a variant of an HIV envelope is employed which is capable of interacting with the CD4-induced antibody in the absence of sCD4. For example, the variant envelope polypeptide could have the first, the second, or both variable loops removed. This variant would expose, create or increase the accessibility of an epitope recognized by a CD4-induced antibody in the absence of sCD4.
As discussed above, the methods and compositions disclosed herein can employ variant polynucleotides and polypeptides of the envelope polypeptide and of the soluble CD4 peptide. As used herein, “variants” is intended to mean substantially similar sequences. A “variant” protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C-terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. As defined herein, the “native” envelope polypeptide of HIV-2 or polynucleotide encoding the same is from the HIV-2 isolate 7312A (SEQ ID NO:2 and 21), the “native” envelope polypeptide of SIVsm or the polynucleotide encoding the same from SIVsmPBj1.9 (SEQ ID NO:3 and 22), the “native” envelope polypeptide of SIVagm or the polynucleotide encoding the same is from SIVagmVer155 (SEQ ID NO:4) and 22 or SIVagmTAN (SEQ ID NO:5 and 24), and the “native” sCD4 polypeptide is set forth in SEQ ID NO:1. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein activity as described herein for envelope and sCD4. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native envelope polypeptide and/or a native soluble CD4 polypeptide employed in the methods of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
A fragment of a biologically active portion of an envelope polypeptide and/or a soluble CD4 polypeptide of the invention will encode at least 15, 25, 30, 50, 100, 150, 200, or 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1,200 contiguous amino acids, or up to the total number of amino acids present in a full-length HIV-2 envelope polypeptide and/or a soluble CD4 polypeptide of the invention.
For polynucleotides, a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the envelope polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode an envelope protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.
Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, an isolated polynucleotide that encodes a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO:21, 22, 23, or 24 are disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
A fragment of an envelope polynucleotide may encode a biologically active portion of an envelope polypeptide. A biologically active portion of an envelope polypeptide can be prepared by isolating a portion of one of the envelope polynucleotide of the invention, expressing the encoded portion of the envelope protein (e.g., by recombinant expression in vitro), and assessing the activity of the portion of the envelope polypeptide. Polynucleotides that are fragments of an envelope nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400 more contiguous nucleotides, or up to the number of nucleotides present in a full-length envelope polynucleotide disclosed herein.
Variant envelope polypeptides and/or a soluble CD4 polypeptide of the invention, as well as polynucleotides encoding these variants, are known in the art and are discussed in further detail elsewhere herein. The polypeptide employed in the methods of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. As discussed below, variant polypeptides or polynucleotides of the invention can comprise heterologous epitopes for HIV-1 binding antibodies. For example, amino acid sequence variants and fragments of the envelope polypeptide and/or a soluble CD4 polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal.
Thus, the polypeptides and polynucleotides employed in the methods of the invention encompass naturally occurring sequences as well as variations and modified forms thereof. Such variants will continue to possess the desired activity for envelope or sCD4 as discussed elsewhere herein. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and optimally will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.
The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated for sCD4 functional variants by the ability to create, expose or render accessible CD4-induced epitopes on the envelope polypeptide. The activity can be evaluated for functional variants of the envelope polypeptides by the ability to interact with CD4 and/or facilitate virus fusion and/or facilitate viral entry into a permissive cell. See, for example, Martin et al (2003) Nature Biotechnology 21:71-76, herein incorporated by reference.
Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSLUM62 scoring matrix; or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
Methods are further provided for a diagnostic assay to monitor HIV-induced disease in a subject and/or to monitor the response of the subject to immunization by an HIV vaccine. By “HIV-induced disease” is intended any disease caused, directly or indirectly, by HIV. An example of an HIV-induced disease is acquired autoimmunodeficiency syndrome (AIDS). The method comprises providing an envelope polypeptide or a functional variant thereof that is not from HIV-1 where the envelope polypeptide further comprises at least one epitope recognized by an HIV-1 binding antibody (i.e., binding, neutralizing, CD4-induced). The envelope polypeptide is contacted with an amount of bodily fluid from the subject; and, the HIV-1 binding antibodies in the bodily fluid of the subject are detected. The detection of the HIV-1 binding antibodies allows the HIV disease in the subject to be monitored. In addition, the detection of the HIV-1 binding antibody also allows the response of the subject to immunization by a HIV vaccine to be monitored. In still other methods, the titer of the HIV-1 binding antibodies is determined. In other methods, the envelope polypeptide is associated with a retrovirus. In this method, a composition comprising a retrovirus having the non-HIV-1 primate lentivirus envelope polypeptide or a functional variant thereof is provided and contacted with the bodily fluid from the subject, and the HIV-1 binding antibodies in the bodily fluid of said patient are detected.
In specific embodiments, the response of the subject to immunization against HIV comprises a 4E10 neutralization response. By “4E10 neutralization response” is intended the increased presence, when compared to an appropriate control, of HIV-1 binding antibodies that interact with the 4E10 epitope. Similarly, a 2F5 or Z13 neutralization response could also be detected.
When a CD4-induced antibody is to be detected, an effective concentration of a soluble CD4/envelope complex which comprises a soluble CD4 or a functional variant thereof and the envelope polypeptide or a functional variant thereof is contacted with an amount of bodily fluid from said subject. The CD4-induced antibodies are detected, and HIV disease in the subject or the response of the subject to immunization by a HIV vaccine is thereby monitored.
Further provided are methods to determine an epitope for an HIV-1 binding antibody. The method comprises providing a population of envelope polypeptides which are not from HIV-1, in which members of the population of the envelope polypeptides comprise at least one epitope recognized by an HIV-1. Members of the population are substantially identical to one another. In specific embodiments, each of the envelope polypeptides in the population is selected from the group consisting of an HIV-2 envelope polypeptide and a functional variant of the HIV-2 envelope polypeptide. In other embodiments, each of the envelope polypeptides in the population is selected from the group consisting of a SIV envelope polypeptide and a functional variant of the SIV envelope polypeptide. The population of envelope polypeptides is contacted with the HIV-1 binding antibody, and the envelope polypeptide or polypeptides in the population that is/are recognized by the HIV-1 binding antibody are determined. The envelope polypeptides in the population can be mixed together and contacted with the HIV-1 binding antibody or alternatively, each envelope polypeptide in the population can be contacted separately by the HIV-1 binding antibody. A comparison of at least one of the amino acid sequences of the envelope polypeptide in the population that binds the HIV-1 antibody with at least one of the amino acid sequences of the envelope polypeptides in the population that do not bind the HIV-1 antibody will allow the epitope for the HIV-1 binding antibody to be determined.
By “substantially identical” is intended the polypeptides in the population have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to one another. Methods to determine percent identity are discussed elsewhere herein. In other embodiments, substantially identical polypeptides will differ by 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids.
Additional methods include an assay to determine the binding characteristics of an HIV-1 binding antibody (i.e., the epitope that the HIV-1 binding antibody interacts with). The method comprises providing an envelope polypeptide or a variant thereof that is not from HIV-1, which comprises an epitope recognized by an HIV-1 binding antibody and contacting the envelope polypeptide with a composition comprising a candidate HIV-1 binding antibody. Assays are performed to determine if the candidate HIV-1 binding antibody recognizes the HIV-1 epitope present in the envelope polypeptide. In this manner, one can characterize the binding properties of the candidate HIV-1 binding antibody. Various candidate HIV-1 binding antibodies are known in the art. Methods are also known to isolate candidate HIV-binding antibodies from a variety of sources including naïve libraries, modified libraries, and libraries produced directly from human donors exhibiting an HIV-specific immune response. See, for example, U.S. Application No. 0030187247.
Methods are also provided to neutralize non-HIV-1 primate lentiviruses, such as HIV-2, SIV, and SRV-1. The method comprises providing a compositions comprising the non-HIV-1 primate lentiviruses and providing an isolated HIV-1 neutralizing antibody. In specific methods, the neutralizing antibody was elicited by HIV-1. In other methods, the neutralizing antibody is from a patient infected with HIV-1. In specific embodiments, the HIV-1 neutralizing antibody is a CD4-induced antibody. In this embodiment, the method comprises providing a composition comprising said HIV-2 or said SIV and providing to the composition an effective concentration of soluble CD4 (sCD4) or a functional variant thereof. An isolated CD4-induced antibody is provided to the composition. Methods to assay for viral neutralization are described elsewhere herein.
Methods are further provided to alter the neutralization potential of a CD4-induced antibody elicited by HIV-1. The method comprises providing a soluble CD4/envelope complex and providing to the soluble CD4/envelope complex a CD4-induced antibody elicited by a HIV-1, and, thereby altering the neutralization potential of the CD4-induced antibody. In still other methods the envelope polypeptide is associated with a retrovirus. In this method, a composition comprising a retrovirus having a non-HIV-1 primate lentivirus envelope polypeptide or a functional variant thereof and an effective concentration of a soluble CD4 polypeptide or a functional variant thereof is provided. A CD4-induced antibody elicited by HIV-1 is also provided to the composition, and the neutralization potential of the CD4-induced antibody is thereby altered. By an “altered” neutralization potential of a CD4-induced antibody is intended any modification (an increase or a decrease) in the ability of the antibody to neutralize a retrovirus having the non-HIV-1 primate lentivirus envelope polypeptide or an active variant thereof when compare to the neutralization activity of the antibody in the absence of soluble CD4 or the functional variant of sCD4. Alteration of neutralization potential can be assayed using the various assays described herein. In specific methods, the sCD4 inducible antibody is from a subject infected with HIV-1.
Further included is a method to identify a soluble CD4 mimic. By “soluble CD4 mimic” is intended any compound that mimics the activity of soluble CD4 (i.e., the compound interacts with the envelope polypeptide or a functional variant thereof, wherein the interaction exposes a CD4-induced epitope on the envelope polypeptide or the functional variant thereof). The compound can include a small inorganic molecule or any organic molecule.
The method comprises providing an envelope polypeptide or a functional variant from a non-HIV-1 lentivirus, contacting the envelope polypeptide or a variant thereof with a candidate compound; and determining if the candidate compound interacts with the envelope polypeptide or functional variant thereof. The interaction of the candidate compound and the envelope polypeptide or functional variant thereof increases the accessibility of an epitope or creates the epitope on the envelope polypeptide or the functional variant thereof. In this method, the created or exposed epitope is recognized by a CD4-induced antibody. Methods of determining whether a particular compound mimics soluble CD4 have been described elsewhere herein. See, also, in Martin et al. (2003) Nature Biotechnology 21:71-76, herein incorporated by reference.
In other methods, the envelope polypeptide is associated with a retrovirus. In this method, a composition comprising a retrovirus having the non-HIV-1 primate lentivirus envelope polypeptide or a functional variant thereof is provided. The retrovirus is contacted with a candidate compound; and it is determined if the candidate compound interacts with the retrovirus. The interaction of the candidate compound and the retrovirus creates, exposes and/or increases the accessibility of a CD4-induced epitope on the envelope polypeptide or the functional variant thereof.
Candidate compounds that may be screened to identify soluble CD4 mimics according to the methods of the invention include any molecule, for example, small inorganic molecules and small organic molecules (e.g., molecules obtained from combinatorial and natural product libraries). Such molecules include, for example, polypeptides (including antibodies and peptides), as well as, nucleic acid molecules, or polysaccharides. It is recognized that the candidate compounds encompass numerous chemical classes.
As will be appreciated by those in the art, candidate compounds can be obtained from a wide variety of sources, including libraries of synthetic and natural compounds. Thus, the methods disclosed herein provide a rapid and easy method for screening any library of candidate compounds. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al (1993) Science 261:1303; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al (1994) J. Med. Chem. 37:1233; and Ghose and Vishwanadhan, eds. (2001) Combinatorial Library Design and Evaluation: Principles, Software Tools, and Applications in Drug Discovery (Marcel Dekker, New York), WO94/24314, and WO94/24314, each of which is herein incorporated by reference in its entirety.
Compositions of the invention include chimeric polypeptides comprising an amino acid sequence encoding an envelope polypeptide or a variant thereof that is not from HIV-1, wherein the amino acid sequence further comprises a heterologous epitope recognized by an HIV-1 binding antibody. In specific embodiments, the epitope recognized by the HIV-binding antibody is a neutralizing HIV-1 epitope, a CD4-induced epitope, or a neutralizing CD4-induced epitope. As used herein, a “heterologous epitope” refers to a domain that is not present in or is found in an alternative location in the native form of the polypeptide or polynucleotide it is contained in. The heterologous epitope can be native to the HIV-1 envelope polypeptide or alternatively, the epitope can be synthetically derived, so long as the epitope continues to be recognized by the HIV-1 binding antibody. Polypeptides or polynucleotides comprising such heterologous epitopes are referred to herein as “chimeric polypeptides” or “chimeric polynucleotides,” respectively. Heterologous epitopes which can be employed in the chimeric polypeptides of the invention are discussed elsewhere herein.
The heterologous epitope or the heterologous domain containing the epitope can be of any length including about 2 to 7 amino acids, about 5 to about 10 amino acids, about 11 to about 20 amino acids, about 21 to about 30 amino acids, about 31 to about 40 amino acids, about 41 to about 50 amino acids, about 51 to about 60 amino acids, about 61 to about 70 amino acids, about 71 amino acids to about 80 amino acids, about 81 to about 90 amino acids, about 91 to about 100 amino acids, about 101 to about 110 amino acids, or longer. The heterologous epitope can be placed anywhere in the envelope sequence, as long as the chimeric polypeptide retains the activity of the envelope polypeptide. Assays to measure envelope activity include, for example, envelope binding assays to CD4, cell fusion assays, and virus entry assays. Such assays are discussed in further detail elsewhere herein. It is recognized that the various methods can be employed to generate the chimeric polypeptide having the heterologous epitope including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
As discussed above, the envelope polypeptide comprising the heterologous epitope may be from any lentivirus that is not HIV-1. Such envelope polypeptides include, but are not limited to, an HIV-2 envelope polypeptide, a functional variant of the HIV-2 envelope polypeptide, a SIV envelope polypeptide, or a functional variant of the SIV envelope polypeptide. Other non-HIV-1 envelope polypeptides are discussed elsewhere herein. Cells and viruses comprising the chimeric polypeptide are encompassed by the invention. In one embodiment, the cell comprising the chimeric polynucleotide or polypeptide comprises a packaging cell line that can be used to generate a viral particle having the chimeric polynucleotide or polypeptide of the invention. Such packaging cell lines are known in the art.
Compositions of the invention further include chimeric polynucleotides. Such chimeric polynucleotides comprises a envelope nucleotide sequence or a variant thereof that is not from HIV-1, wherein the nucleotide sequence further comprises a heterologous epitope encoding an epitope recognized by an HIV-1 binding antibody. In specific embodiments, the heterologous epitope recognized by the HIV-binding antibody is a neutralizing HIV-1 epitope, a CD4-induced epitope, or a neutralizing CD4-induced epitope. Cells and viruses comprising the chimeric polypeptide are further provided.
The nucleotide sequence encoding the heterologous epitope or the domain it is contained in can be of any length including about 15 to about 30 nucleotides, about 31 to about 60 nucleotides, about 61 to about 90 nucleotides, about 91 to about 120 nucleotides, about 121 to about 150 nucleotides, about 151 to about 180 nucleotides, about 181 to about 210 nucleotides, about 210 to about 240 nucleotides, about 241 to about 270, about 271 to about 300, about 301 to about 330 nucleotides, or longer. It is recognized that the various methods can be employed to generate the chimeric polynucleotide having the heterologous epitope including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
The use of the term “polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides, can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. Methods of generating such sequences are discussed elsewhere herein.
Exemplary chimeric polynucleotides and polypeptides of the invention include sequences encoding non-HIV-1 envelope polypeptides, or variants thereof, which have been modified to have an HIV-1 MPER region, a 4E10, a Z13, or a 2F5 epitope or functional variants (immunologically equivalent epitopes) are discussed elsewhere herein. Non-limiting examples of such chimeric polynucleotides and polypeptides include the envelope polypeptide of HIV-2 7312A in which amino acids 675 and 676 (HXB-2c numbering system) are altered from L to I and from A to T, respectively. As shown in
Additional non-limiting examples include the envelope polypeptide of HIV-2 7312A or HIV-2 ST in which the 2F5 epitope, or the immunologically equivalent epitope thereof, is engineered into the polynucleotide. One such chimeric polypeptide, and the chimeric polynucleotide encoding it includes the polypeptide having site-directed mutations in the HIV-2 7312A envelope polypeptide at positions 660 (K to A), 662 (N to D), 663 (S to K), and 665 (D to A) of SEQ ID NO:2, which together make the HIV-2 sequence identical to that of the 2F5 epitope region of HIV-1 YU2. As shown in
The chimeric polynucleotide of the invention can be provided in expression cassettes for expression in a cell of interest. The cassette can include 5′ and 3′ regulatory sequences operably linked to the chimeric polynucleotide of the invention. “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a chimeric polynucleotide of interest and a regulatory sequence (i.e., a promoter) is functional link that allows for expression of the chimeric polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the cell of interest. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the chimeric polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
The expression cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a chimeric polynucleotide of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in the cell type of interest. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the chimeric polynucleotide of the invention may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the chimeric polynucleotide of the invention may be heterologous to the host cell or to each other.
In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
Additional compositions of the invention comprise kits comprising a retrovirus having the envelope polypeptide or a functional variant thereof from a non-HIV-1 primate lentivirus. Additional compositions comprise kits comprising the retrovirus having the envelope polypeptide or a functional variant thereof from the non-HIV-1 primate lentivirus along with sCD4 of a functional variant thereof. Kits of the invention can also comprise the chimeric polypeptides and polynucleotides described herein. Any kit can further be accompanied by instructions for use as discussed elsewhere herein.
The following examples are offered by way of illustration and not by way of limitation.
In the present study, we sought to identify immunogenic, broadly-cross reactive epitopes on the HIV-1 envelope glycoprotein that might serve as targets of the adaptive humoral immune response in naturally-infected humans. We hypothesized that conserved requirements for co-receptor binding among diverse lineages of human or simian immunodeficiency viruses might be reflected in conserved antigenicity at the corresponding envelope surface. As a strategy, we took advantage of the wide evolutionary distance that exists between HIV-1 and HIV-2 lineages to probe for conserved neutralization epitopes. The envelope glycoproteins of HIV-1 and HIV-2 are only about 40% homologous in amino acid sequence (HIV Sequence Compendium 2002. Kuiken et al. Eds. Los Alamos National Laboratory, Los Alamos, N. Mex., LA-UR 03-3564). As a consequence, they generally exhibit weak antigenic cross-reactivity, and sera from HIV-1 infected individuals cross-neutralize HIV-2 poorly if at all (Weiss et al. (1988) Aids 2:95-100; Bottiger et al. (1990) J Virol 64:3492-3499; Thomas et al. (2003) AIDS 17:291-300). Nonetheless, HIV-1 and HIV-2 each require chemokine co-receptor binding for cell entry, with primary non-T cell line adapted viruses of both types generally utilizing CCR5 (Deng et al. (1997) Nature 388:296-300; Zhang et al. (2000) J Virol 74:6893-6910). Binding of CD4 to HIV-1 gp120 induces conformational changes in the outer and inner envelope domains, the bridging sheet, and the positioning of variable loops V1/V2 and V3 (Sattentau et al. (1993) J Virol 67:7383-7393; Wu et al. (1996) Nature 384:179-183; Trkola et al. (1996) Nature 384:184-187; Salzwedel et al. (2000) J Virol 74:326-333; Rizzuto et al. (1998) Science 280:1949-1953; Rizzuto (2000) AIDS Res Hum Retroviruses 16:741-749). These changes lead to exposure of the envelope co-receptor binding site, comprised of the bridging sheet, adjacent surfaces, and possibly the tip of V3. Antibodies that bind to HIV-1 gp120 preferentially (or only) after CD4 engagement are referred to as CD4-induced (CD41). Typically, these antibodies bind to surfaces that include or are proximal to the bridging sheet where they compete with co-receptor binding and broadly (but not potently) neutralize different HIV-1 strains (Salzwedel et al (2000) J Virol 74:326-333; Rizzuto et al. (1998) Science 280:1949-1953; Rizzuto (2000) AIDS Res Hum Retroviruses 16:741-749; Xiang et al. (2002) AIDS Res Hum Retroviruses 18:1207-1217; Xiang et al. (2003) Virology 315:124-134; Huang et al. (2004) Proc Natl Acad Sci USA 101:2706-2711). Cross-reactivity between HIV-1 induced CD4i antibodies and HIV-2 has not been reported. Here, we explore the antigenic cross-reactivity and inherent immunogenicity of the co-receptor binding surfaces of HIV-1 and HIV-2 and assess whether HIV-2, in complex with sCD4, might be useful as a specific probe for HIV-1 elicited CD4i neutralizing antibodies in humans infected by HIV-1 or immunized with candidate HIV-1 vaccines.
Materials and Methods
Plasma Specimens. Pre-existing coded plasma samples from 189 HIV-1 infected subjects and 15 uninfected normal control individuals were analyzed. Blood was generally collected in acid citrate dextrose, platelet-free plasma prepared by sequential 10 min centrifugations at 200 g and 1000 g, and 1 ml aliquots stored at −20° C. or −70° C. Prior to use, plasma was thawed, heat-inactivated at 56° C. for 30 min, and clarified by centrifugation at 3000 g for 5 min. Human subjects gave informed consent and protocols received institutional review board approvals.
Cell Entry and Neutralization Assays. Plasma samples and monoclonal antibodies were assayed for Nab activity using a modification of a recently described HIV entry assay (3) that employs the surface adherent HeLa cell-derived JC53BL-13 cell line (NIH AIDS Research and Reference Reagent Program catalogue #8129, TZM-b1), which has been genetically-modified and selected so as to constitutively express CD4, CCR5 and CXCR4. The cells contain integrated luciferase and β-galactosidase (P-gal) genes under tight regulatory control of an HIV-1 LTR. Virus stocks were obtained by transfection of 293T cells and were titered by β-gal expression on JC53BL-13 cells, as described (Wei et al. (2003) Nature 422:307-312). 7×103 JC53BL-13 cells were plated in 96-well tissue culture plates (Falcon) and cultured overnight in DMEM supplemented with 10% fetal calf serum (FCS). 3,000 infectious units of virus were combined in a total volume of 60 μl with or without a 2× concentration of sCD4 in DMEM with 6% FCS and 80 ug/ml DEAE-dextran. After 1 hr at 37° C., an equal volume of test or control plasma (10% vol/vol in DMEM plus 6% FCS or five-fold dilutions thereof) or monoclonal antibody was added. This brought the final concentration of DEAE dextran to 40 μg/ml and that of human plasma to 5%. It is important to note that sufficient normal human plasma (NHP) was added to each well so as to maintain a constant final human plasma concentration of 5% in each virus +sCD4+ test plasma mixture. Concentrations of NHP (or test plasma) that exceed 5% commonly result in nonspecific inhibition of virus entry (Wei et al. (2003) Nature 422:307-312), and thus samples are not tested for neutralizing activity at dilutions less than 1:20. The concentration of sCD4 was chosen so that the final 1× concentration after the addition of test plasma corresponds to the IC50 of sCD4 specific for each virus. The virus +sCD4+ test plasma (or monoclonal antibody) mixture was incubated for 1 hr at 37° C. Media was removed entirely from the adherent JC53BL-13 monolayer just before the addition of the virus +sCD4+ test plasma (or monoclonal antibody) to it. Cells were incubated at 37° C. for 2 days and then analyzed for luciferase expression, as described (Wei et al. (2003) Nature 422:307-312). Controls included cells exposed to no virus and to virus pretreated with NHP or control monoclonal antibodies only. Relative infectivity was calculated by dividing the number of luciferase units at each dilution of test plasma or monoclonal antibodies by values in wells containing NHP but no test plasma or monoclonal antibodies. Neutralization was assessed by 50% inhibitory concentration (IC50) determined by linear regression using a least-squares method. All samples were tested in duplicate and all experiments repeated at least three times to ensure reproducibility.
A Cf2Th-synCCR5 cell assay was used to test viruses for CD4-independent cell fusion and entry. Envelope glycoproteins from plasma derived virion RNA/cDNA were expressed in 293T cells and used to pseudotype an env-defective HIV-1 reporter virus (pNLENG1-ES-IRES) containing an enhanced green fluorescent protein (GFP) gene (Levy et al. (2004) Proc Natl Acad Sci USA 101:4204-4209). Infectious titers of pseudotyped virus were determined first in JC53BL-53 cells so that virus inoculae could be standardized. Cf2Th-synCCR5 cells (Mirzabekov et al. (1999) J Biol Chem 274:28745-28750), which express human CCR5 but not CD4, were plated in 24-well tissue culture plates at a density of 4×104 cells/well and cultured overnight in standard medium (90% DMEM, 10% FBS, 0.5 mg/ml G418, 3.0 ug/ml puromycin, 100 units/ml penicillin, and 100 ug/ml streptomycin) at 37° C. and 5% CO2. Virus, with or without pre-incubation with sCD4, and with or without pre-incubation with monoclonal antibodies or test plasma, was then added in a total volume of 0.25 ml of standard culture medium and incubated for 5 hours at 37° C. If neutralization assays were performed with human plasma, attention was again given to ensure that 5% vol/vol total concentration of plasma was maintained in all wells, as described above in the JC53BL-13 assay. An additional 0.25 ml of medium was then added and the cultures were maintained for 48 hours at 37° C. Thereafter, cells were washed in PBS and visualized directly for GFP expression or detached from the plates by trypsin-EDTA, collected in a 2 ml eppendorf tube, and washed once with PBS before resuspension in 0.3 ml PBS. GFP positive cells were then determined by FACS analysis (Mirzabekov et al. (1999) J Biol Chem 274:28745-28750). To test for CCR5-dependent, CD4-independent envelope-mediated fusion, the assay was modified by omitting the env-defective HIV-1 reporter virus (pNLENG1-ES-IRES) and quantifying syncytium formation resulting from co-culture of env-expressing 293T cells and Cf2Th-synCCR5 cells.
Virus stocks. For neutralization experiments in JC53BL-13 cells, HIV-2 proviral clones pJK7312A (GENBANK #L36874) (36-38), pJK7312A/V434M, pJK7312A/H419R, and pJK7312A/Q422L, each cloned in pBlueScript II SK at NotI/EcoRI sites, and pJSP4-27(ST/SXB1) (Deng et al. (1997) Nature 388:296-300; Kumar et al. (1990) J Virol 64:890-901), were used to transfect 293T cells. HIV-2 UC-1 env (Deng et al. (1997) Nature 388:296-300; Barnett et al. (1993) J Virol 67:1006-1014) and HIV-1133M env, cloned in pSM and pCR3.1, respectively, were co-transfected with pSG3deltaEnv or pJK7312AdeltaEnv to create infectious pseudovirions, as described (Wei et al. (2003) Nature 422:307-312). For cell entry experiments using Cf2Th-synCCR5 cells (35), HIV-1 env genes cloned in pcDNA3.1 were co-transfected with an HIV-1 reporter virus (pNLENG1-ES-IRES) that contains an enhanced green fluorescence gene (Mirzabekov et al. (1999) J Biol Chem 274:28745-28750) using the FuGENE 6 transfection kit (Roche Diagnostics). For antibody binding studies, HIV and SIV envelope glycoproteins were obtained from 293T cells transfected with HIV-27312A; MT4 cells infected by HIV-2MVP15132 (Beyl et al. (1987) Munch Med Wochenschr 129:895-896; Gao et al. (1993) AIDS Res. Hum Retroviruses 9:703-704), HIV-2CBL20 (Schulz et al. (1990) J Virol 64:5177-5182), or SIVmac239; and 293T cells infected with recombinant vaccinia viruses expressing HIV-1 JR-FL, HIV-1 Ba-L, or SIVmne gp160 genes.
Binding and Competition Assays. Biotinylated monoclonal antibodies were tested for binding to HIV-2, SIV or HIV-1 gp120 envelope glycoproteins captured in wells of microtiter plates coated with Mab 2.6C or EH21, as previously described (Xiang et al. (2002) AIDS Res Hum Retroviruses 18:1207-1217; Xiang et al. (2003) Virology 315:124-134). Prior to the addition of biotin-labeled antibodies, gp120 was pre-incubated with sCD4 (R&D Systems, catalogue #514D; 1-10 ug/ml) or a mock preparation. Binding was quantified by the reaction of peroxidase conjugated streptavidin and subsequent color development with substrate TMB-H2O2. Competition assays were performed by preincubating plasma samples with immobilized gp120-sCD4 complexes and then determining binding of biotin-labeled Mabs at subsaturating concentrations, as described (Xiang et al. (2002) AIDS Res Hum Retroviruses 18:1207-1217; Xiang et al. (2003) Virology 315:124-134).
Monoclonal antibodies. The prototypic CD4i monoclonal antibodies (Mabs) 17b and 48d, and several more recent CD4i Mabs, 23e, 21c, 4.11 g, 412d, E51 and CM51, have been described (Xiang et al. (2003) Virology 315:124-134; Huang et al. (2004) Proc Natl Acad Sci USA 101:2706-2711; Thali et al. (1993) J Virol 67:3978-3988; Choe et al. (2003) Cell 114:161-170; HIV Immunology and HIV/SIV Vaccine Databases 2003. Korber et al. Eds. Los Alamos National Laboratory, N. Mex. LA-UR 04-8162). Additional CD4i Mabs used in this study were isolated from HIV-1 infected subjects started on HAART during acute infection. These include 19e, ED47, ED49, ED10, ED11, 31H, 58H and 28d. All of the CD4i Mabs bind to the HIV-1 gp120 glycoprotein co-receptor binding surface that is created (or exposed) following sCD4 binding or deletion or repositioning of V1/V2 variable loop sequences. But three of the Mabs, 19e, ED47 and ED49, are unusual in that they bind poorly, or not at all, to V1/V2 deleted HIV-1 gp120. Hence, their binding is CD4-dependent. Further characteristics of these Mabs will be presented in a separate publication. The other Mabs specific for the HIV-1 CD4 binding site, variable loops, surface glycans, and other gp120 and gp41 epitopes have been described (HIV Immunology and HIV/SIV Vaccine Databases 2003. Korber et al. Eds. Los Alamos National Laboratory, N. Mex. LA-UR 04-8162). Human Mabs 1.7 and 2.6C have specificity for HIV-2 gp120 and were isolated from an HIV-2 infected West African patient, as previously described (Cole et al. (2001) Virology 290:59-73; Robinson et al. (1998) AIDS Res Hum Retroviruses 14:1253-1262). The anti-CD4 Mab from clone RPA-T4 was obtained from BD Biosciences (catalogue # 555344).
Molecular Cloning, Sequencing, and Mutagenesis. Full length gp160 envelope genes were amplified by nested PCR from plasma HIV-1 RNA. Virion-associated plasma RNA was prepared using the QIAmp Viral RNA Mini Kit (Qiagen) as previously described (Wei et al. (2003) Nature 422:307-312; Wei et al. (1995) Nature 373:117-122). From each timepoint, replicate plasma virus RNA preparations (4000-8000 RNA molecules per reaction) were subjected to cDNA synthesis using SuperScript II (Invitrogen). Replicate viral cDNA samples (1, 10, 100, or 1000 molecules each) were then subjected to nested PCR amplification as described, using the following primers: Outer sense primer (5′-TAGAGCCCTGGAAGCATCCAGGAAG-3′, nt 5852-5876) (SEQ ID NO:17), outer anti-sense primer (5′-TTGCTACTTGTGATTGCTCCATGT-3′, nt 8912-8935) (SEQ ID NO: 18), inner sense primer (5′-GATCAAGCTTTAGGCATCTCCTATGGCAGG AAGAAG-3′, nt 5957-5982) (SEQ ID NO: 19), and inner anti-sense primer (5′-AGCTGGATCCGTCTCGA GATACTGCTCCCACCC-3′, nt 8881-8903) (SEQ ID NO: 20). Inner primers contain additional 5′ sequences and restriction sites to facilitate cloning. The PCR products of the full-length env genes were cloned into pcDNA3.1 (Invitrogen) for expression. All clones, including those modified by site-directed mutagenesis, were sequenced using an ABI 3100 Genetic Analyzer and dideoxy methodology. Sequences have been deposited in GENBANK (accession numbers AY223761-90; AY223720-54; additional entries pending). To ensure that molecular clones of HIV-1 envelope amplified from plasma viral RNA were representative of plasma virus, replicate PCR reactions were performed on primary samples at varying endpoint titrations of viral cDNA and on separate days. Site-directed mutagenesis was done using the Quik-Change™ site-directed mutagenesis kit (Stratagene Inc.). 125 ng of complementary primers with mutant sequences and 20 ng of template pcDNA3.1-env were used for each PCR amplification. PCR conditions were as follows: 95° C. for 50 sec, 60° C. for 50 sec, and 68° C. for 10 min. After 16 cycles the PCR product was digested with 10 units of DpnI to cleave template DNA at 37° C. for 1 hr. Mutants were identified and confirmed by nucleotide sequencing.
Statistical Analyses. Linear regression, Pearson correlations, Fisher's exact test, and Wilcoxon rank sum test were performed on primary and log transformed data sets. Calculations were performed in SAS.
Supplementary Material.
Results
Plasma from HIV-1 Infected Patients Neutralizes CD4-induced HIV-2. Table 1 shows the extent and kinetics of the Nab response to autologous HIV-1 virus in a patient (133M) following subtype C HIV-1 infection.
aThe HIV-1 gp160 env gene from patient 133M was PCR amplified and cloned from uncultured month 2 peripheral blood mononuclear cells and used to prepare pseudotyped virus.
bReciprocal IC50 titer of neutralizing antibodies as determined in JC53BL-13 cells (1).
Nab titers against the earliest detectable virus reached 1:2,500 (50% inhibitory concentration, IC50) by 11 months of infection and then subsided. Such a response is typical of patients with newly acquired HIV-1 infection, and it is generally followed rapidly by virus mutation and escape from neutralization (Wei et al. (2003) Nature 422:307-312; Richmanetal. (2003) Proc Natl Acad Sci USA 100:4144-4149). To look for more broadly reactive Nabs in this subject, we applied these same plasma specimens to the HIV-2 strain 7312A, a primary CD4-dependent R5 virus (Deng et al. (1997) Nature 388:296-300; Zhang et al. (2000) J Virol 74:6893-6910; Deng et al. (1997) Nature 388:296-300; Zhang et al. (2000) J Virol 74:6893-6910). As expected, plasma from this HIV-1 infected patient (133M) exhibited no detectable neutralizing activity against HIV-27312A, a finding consistent with prior studies showing little neutralization cross-reactivity between these highly divergent viral lineages (Weiss et al (1988) Aids 2:95-100; Bottiger et al (1990) J Virol 64:3492-3499). However, when HIV-27312A was pretreated for 1 hour with 9 nM sCD4 (equal to the IC50 for this virus), the virus became remarkably susceptible to neutralization by 133M plasma, with titers of Nab reaching 1:12,500 by 26 months following infection (Table 1). Similar results were obtained in six additional subjects with primary subtype C HIV-1 infection whose Nab titers to sCD4-pretreated HIV-27312A ranged from 1:53 to 1:3,361 and which peaked between 8 and 24 months following acute infection. To determine if the CD4-dependent Nab activity that we observed in plasma from subtype C patients was limited to this virus lade, we studied additional patients chronically infected with HIV-1 subtypes A, B, C or D.
HIV-1 CD4i Monoclonal Antibodies Neutralize CD4-induced HIV-2. If the broadly cross-reactive neutralizing antibody activity that we observed in HIV-1 infected patient plasma is due to classical CD4i antibodies, then prototypic CD41 monoclonal antibodies derived from HIV-1 infected patients, which have been extensively characterized against HIV-1 envelope glycoproteins (Salzwedel et al. (2000) J Virol 74:326-333; Rizzuto et al. (1998) Science 280:1949-1953; Rizzuto (2000) AIDS Res Hum Retroviruses 16:741-749; Xiang et al. (2002) AIDS Res Hum Retroviruses 18:1207-1217; Xiang et al. (2003) Virology 315:124-134; Huang et al. (2004) Proc Natl Acad Sci USA 101:2706-2711), might be expected to cross-neutralize HIV-2 in a CD4-dependent fashion.
Multiple Primary HIV-2 Strains are Susceptible to HIV-1 CD4i Antibody Neutralization. Neutralization of HIV-2 by HIV-1 elicited CD4i antibodies is not restricted to HIV-27312A and derivative strains. HIV-2UC-1 and HIV-2ST/SXB1, two other well-characterized HIV-2 R5-tropic viruses (Deng et al. (1997) Nature 388:296-300; Barnett et al. (1993) J Virol 67:1006-1014), also demonstrated striking neutralization susceptibility to HIV-1 elicited CD4i monoclonal antibodies and to HIV-1 infected patient plasma in patterns that were similar (but not identical) to HIV-27312A. Results for HIV-27312A and HIV-2UC-1 are compared in Table 2.
aValues preceding toe slash marks denote the IC50 in μg/ml for monoclonal antibodies and in reciprocal dilutions for patient plasma specimens, each in the absence of sCD4. Values following the slash marks denote IC50 values in the presence of sCD4. sCD4 concentrations were adjusted to correspond to the IC50 specific
n.d., not done.
Each virus was susceptible to 21c and 19e and to a lesser extent 17b, 31H and ED49. HIV-2UC-1 was more susceptible to E51 and 31H, but much less susceptible to 17b, compared with HIV-27312A. Both viruses were completely resistant to neutralization by 13 different HIV-1 elicited non-CD4i human monoclonal antibodies, including those targeting the CD4 binding site (CD4bs), V3 loop, surface glycans, and gp41. HIV-2UC-1 was also compared with HIV-27312A in its susceptibility to neutralization by a subset of ten HIV-1 clade A, B, C and D patient plasmas (Table 2, bottom). CD4-dependent Nab titers against HIV-2UC-1 were at least two-fold higher than for HIV-27312A in two patients (6X4F and 21X0F), three-fold lower in one patient (37X4F), and not substantially different in seven others. For each HIV-1 antibody positive plasma specimen tested, there was a one to three log CD4-dependent shift in the HIV-2UC-1 neutralization curve (Table 2, bottom).
HIV-1 CD4i Antibody Binding to HIV-2 Glycoprotein Correlates With Neutralization. CD4i antibodies in HIV-1 plasma that neutralize HIV-2 infection might also be expected to compete directly with HIV-1 CD4i monoclonal antibodies for binding to HIV-2 gp120-sCD4 complexes.
To further examine the correlation between antibody binding and neutralization, we tested a large number of biotin-labeled HIV-1 CD4i antibodies for binding to HIV-27312A envelope glycoprotein with and without sCD4.
Site-directed Mutagenesis of the HIV-2 Bridging Street Alters HIV-1 CD4i Antibody Recognition. HIV-2 neutralization by HIV-1 CD4i monoclonal and polyclonal antibodies is best explained by antibodies binding to the conserved chemokine co-receptor binding surface, including the bridging sheet. To evaluate this hypothesis directly, we performed site-directed mutagenesis on the HIV-2 bridging sheet region (Reeves et al. (2002) J Gen Virol 83:1253-1265). The primary amino acid sequence of the bridging sheet of HIV-1 and the corresponding sequence of HIV-2 is conserved but not identical (
Prevalence and Titers of CD4i Neutralizing Antibodies in Patients Infected by Diverse HIV-1 Subtypes. Plasma samples from 189 individuals infected by HIV-1 clades A, B, C, D, F, G or H, or by CRF01, CRF02 or CRF11, were tested for CD4i Nabs against HIV-2. In preliminary studies, we tested a subset of 69 of these specimens for reactivity against the wildtype HIV-2 strain 7312A and its derivative 7312A/V434M. This pilot study showed that the frequency of detection of HIV-2 cross-reactive CD4i Nabs was modestly higher for the V434M virus (94%) compared with 7312A (87%). Based on the enhanced sensitivity of HIV-27312A/V434M, we used this virus to test all 189 patient plasma specimens for CD4i Nabs (Table 3).
aReciprocal IC50 titers of CD4i neutralizing antibodies against HIV-27312A/V434M pretreated with 15 nM sCD4.
CD4i Nabs were detected in 174 (92%) of patients, with median IC50 titers of 0.0004 (1:2,500) and mean titers of 0.004 (1:250). Titers of CD4i Nab in plasma from clade D and CRF11 patients, considered separately or as a group, were significantly greater than for patients in the remaining groups (p<0.0001). We considered the possibility that, despite the overall similarity in neutralization patterns observed for the HIV-2 strains depicted in Table 2, divergent HIV-2 strains might detect CD4i Nabs in some of the patient's plasmas that tested negative against HIV-27312A/V434M. Thus, we retested the 15 negative samples, first by western immunoblot to confirm HIV-1 positivity, and then by neutralization assay against two different HIV-2 strains: UC-1, ST/SXB1, and 7312A. All 15 samples were western immunoblot positive against HIV-1 proteins. Four samples were found to have CD4i Nabs against one or more of these viruses in titers ranging from 1:25 to 1:750. Thus, overall, out of 189 HIV-1 infected patients tested, 178 (94%) had detectible neutralizing CD4i antibodies against HIV-2.
Role of CD4i Antibodies in Natural HIV-1 Infection. Previous studies have shown that HIV-1 CD4i antibodies are largely excluded by steric hindrance from the virus:cell interface following CD4 engagement, and as a consequence, CD41 antibodies generally neutralize HIV-1 inefficiently (Labrijn et al. (2003) J Virol 77:10557-10565; Salzwedel et al. (2000) J Virol 74:326-333). However, this steric restriction could be overcome experimentally by using CD4i antibody fragments (Fab or sFv) or by disassociating (spatially or temporally) envelope-CD4 engagement from envelope-coreceptor engagement (Labrijn et al. (2003) J Virol 77:10557-10565; Salzwedel et al. (2000) J Virol 74:326-333). Given these constraints on CD4i antibody-mediated neutralization, we sought to examine what role CD4i antibodies might play in vivo. Sodroski and colleagues (Kolchinsky et al (2001) J Virol 75:2041-2050) first postulated that CD4i antibodies might constrain virus to CD4 dependence by selecting against envelope mutations that lead to spontaneous exposure of the viral co-receptor binding surface (Kolchinsky et al (1999) J Virol 73:8120-8126; Hoffman et al. (1999) Proc Natl Acad Sci USA 96:6359-6364). Our results support this hypothesis by showing in naturally-infected humans that CD4i antibodies are prevalent, high-titer, and so broadly cross-reactive that they neutralize even HIV-2. However, to test more directly if CD4i antibodies might be active in constraining HIV-1 to CD4 dependence in vivo, we examined sequential uncultured plasma specimens from four HIV-1 infected patients (133M, WEAU0575, SUMA0874, BORI0637) for evidence of viruses that contain mutations in envelope that result in greater spontaneous exposure of the receptor binding surfaces. Seventy-four full-length, functional gp160 envelope clones were derived by polymerase chain reaction (PCR) amplification of plasma virion RNA and used to pseudotype env-deficient HIV-1 virus for entry in JC53BL-13 cells. Two clones from patient SUMA0874 (S736-68 and S736-75) were found to be uniquely sensitive to neutralization by sCD4 (IC50<0.05 ug/ml), indicating that they might exhibit greater spontaneous exposure of receptor binding surfaces than is generally observed in primary HIV-1 strains (Pugach et al. (2004) Virology 321:8-22). These same two clones were also distinguished from all others that we examined by an isoleucine (I) to threonine (T) substitution at position 309 (HXB2 numbering system) immediately 5′ of the GPGR crown of the V3 loop (
Discussion
Although much is already known about the structure, function, and antigenic properties of the HIV-1 envelope glycoprotein (Parren et al. (1999) Aids 13 Suppl A:S137-162; Kwong et al. (1998) Nature 393:648-659; Wyatt et al. (1998) Nature 393:705-711; Wyatt et al. (1998) Science 280:1884-1888; Kwong et al. (2002) Nature 420:678-682; Labrijn et al. (2003) J Virol 77:10557-10565; Burton et al. (2004) Nat Immunol 5:233-236; Zolla-Pazner et al (2004) Nat Rev Immunol 4:199-210; Broliden et al. (1992) Proc Natl Acad Sci USA 89:461-465; Scala et al. (1999) J Immunol 162:6155-6161; Opalka et al. (2004) J Immunol Methods 287:49-65; Sattentau et al. (1993) J Virol 67:7383-7393; Wu et al. (1996) Nature 384:179-183; Trkola et al. (1996) Nature 384:184-187; Salzwedel et al. (2000) J Virol 74:326-333; Rizzuto et al. (1998) Science 280:1949-1953; Rizzuto (2000) AIDS Res Hum Retroviruses 16:741-749; Xiang et al. (2002) AIDS Res Hum Retroviruses 18:1207-1217; Xiang et al. (2003) Virology 315:124-134; Huang et al. (2004) Proc Natl Acad Sci USA 101:2706-2711), the present study provides new insight into the immunogenicity and antigenic conservation of the envelope co-receptor binding site in natural human infection and the likely biological role of CD4i antibodies elicited against it. Previous studies, based largely on the identification and characterization of HIV-1 specific human monoclonal antibodies, suggested that the conformationally-dependent co-receptor binding surface on HIV-1 was only weakly immunogenic and CD41 antibodies relatively uncommon (Xiang et al. (2002) AIDS Res Hum Retroviruses 18:1207-1217; Xiang et al (2003) Virology 315:124-134; Huang et al. (2004) Proc Natl Acad Sci USA 101:2706-2711). However, the recent identification of increasing numbers of CD4i monoclonal antibodies from patients with acute and early HIV-1 infection (J.E.R., unpublished), together with findings described in this report, indicate quite the opposite to be the case. We find the vast majority (94%) of HIV-1 infected patients, infected by any one of ten different clades or CRFs, harbor HIV-specific CD4i Nabs with IC50 titers ranging from 1:20 to greater than 1:100,000. The mean CD4i Nab titer against HIV-27312/V434M among 189 subjects was 1:250 and the median titer 1:2,500. 114 subjects had Nab titers equal to or greater than 1:1,000, the highest reaching 1:143,000. Of interest, patients with subtype D and CRF11 infection had statistically higher titers of CD4i Nabs than did other individuals (p<0.0001). In a related study, we found that 8 of 10 healthy, uninfected human volunteers who were immunized with ALVAC vCP1452 HIV-1 gp140 alone or in combination with soluble monomeric HIV-1 gp120 (AIDSVAX B/B), developed HIV-1 CD41 neutralizing antibodies against HIV-27312A, compared with none of 5 control subjects who were vaccinated with placebo (J.M.D. and G.M.S., manuscript in preparation). To explain the elicitation of CD4i Nabs by soluble HIV-1 gp120 or expressed gp140, we suspect that envelope glycoprotein is bound to cell-surface-associated CD4, undergoes conformational change, and elicits a CD4i antibody response. Regardless of the mechanism, it is clear from our studies that the co-receptor binding site of the HIV-1 glycoprotein presented either in the context of natural infection or by vaccination with expressed or soluble glycoprotein, is inherently immunogenic and neutralization of sCD4-triggered HIV-2 is a sensitive and specific means for detecting these CD4-induced antibodies.
The observation that CD4i antibodies elicited by HIV-1 infection potently neutralized multiple strains of HIV-2 came as a surprise. While most primary human and simian lentiviruses use CCR5 as a co-receptor for cell attachment and entry (Zhang et al. (2000) J Virol 74:6893-6910), functionally important amino acids in the HIV-1 envelope co-receptor binding region identified by mutagenesis experiments (Rizzuto et al. (1998) Science 280:1949-1953; Rizzuto (2000) AIDS Res Hum Retroviruses 16:741-749; Kwong et al (1998) Nature 393:648-659) are only partially conserved in HIV-2, SIVmac and SIVagm (see
It is of interest to consider the cooperative interactions that may be occurring among sCD4, the HIV-2 envelope glycoprotein, and CD4i antibody that result in potent virus neutralization. We have ruled out the possibility that HIV-1 elicited CD4i antibodies neutralize HIV-2 by binding directly to CD4, since a scorpion toxin-based CD4 mimetic that differs substantially in amino acid sequence from CD4 also results in conformational changes in HIV-2 gp120 leading to binding and neutralization by different monoclonal and polyclonal CD4i antibodies (J.M.D., P.D.K., J.A.R., G.M.S., unpublished). Moreover, the contact residues of several of the HIV-1 CD4i monoclonal antibodies that cross-neutralize HIV-2 have been resolved within antibody: HIV-1 gp120: sCD4 complexes, and they do not include contact points on CD4 (Kwong et al. (1998) Nature 393:648-659; Rizzuto (2000) AIDS Res Hum Retroviruses 16:741-749; Xiang et al. (2002) AIDS Res Hum Retroviruses 18:1207-1217; Darbha et al. (2004) Biochemistry 43:1410-1417). If sCD4 does not interact directly with CD4i antibodies in the context of the envelope trimer, then it must enhance the susceptibility of virus to neutralization by inducing conformational change and exposure of CD4i epitopes, but in a cooperative manner, since the magnitude of HIV-2 neutralization we observe is far greater than would be expected on the basis of additive stoichiometry. For example, in the CD4i Nab assay, we routinely use a concentration of sCD4 equal to its IC50 for each virus (e.g., 9 nM for HIV-27312, 3 nM for HIV-2UC-1, and 25 nM for HIV-2ST/SXB1). This concentration of sCD4, by definition, reduces the number of infectious units (i.u.) measured by 50%, for example from 10,000 i.u. to 5,000 i.u., which corresponds to 100% infectivity (see
The role that CD4i antibodies play in natural HIV-1 infection is becoming more clear. Our data, together with other results (Kolchinsky et al. (2001) J Virol 75:2041-2050; Zhang et al. (2002) J Virol 76:644-655), indicate that spontaneously-occurring HIV-1 variants that exhibit an exposed co-receptor binding surface and CD4 independence, are generated in vivo where they are almost certainly targeted for neutralization by CD4i or other HIV-1 specific antibodies. In fact, four studies have now shown that single amino acid substitutions in the HIV-1 glycoprotein, either at the base of V1/V2 (Kolchinsky et al. (2001) J Virol 75:3435-3443; Wei et al. (2003) Nature 422:307-312) or in the V3 loop (Zhang et al. (2002) J Virol 76:644-655 and this report), are sufficient to confer on the virus varying degrees of CD4 independence, greater spontaneous exposure of the co-receptor binding site, and enhanced susceptibility to CD4i Nabs. Principles of viral dynamics, coupled with the well documented error-prone nature of HIV-1 reverse transcriptase, indicate that such mutations must be occurring in vivo on a virtually continuous basis, as has been documented for comparable mutations leading to anti-retroviral drug resistance (Wei et al. (1995) Nature 373:117-122). Thus, CD4i antibodies may influence HIV-1 natural history and pathogenesis to a greater extent than is currently recognized by limiting the spectrum of cells available as targets of virus infection to those expressing surface CD4. In this context, three observations are of note: First, Gabuzda and colleagues have reported that HIV-1 virus within the central nervous system sanctuary (where circulating antibodies are relatively excluded) has less dependence on cell surface bound CD4 for its attachment and entry and such viruses may target CD4-negative astrocytes as well as CD4lo microglial cells for infection (Gorry et al. (2002) J Virol 76:6277-6292). Secondly, the three HIV-2 virus strains that we found to be susceptible to HIV-1 CD4i antibody neutralization (7312A, UC-1, ST/SXB1) all utilize CCR5 as a co-receptor, whereas three other HIV-2 strains (UC-2, ROD-B, MVP15132) that we examined utilize X4 for cell entry and were not susceptible to HIV-1 CD4i antibody neutralization. Interestingly, monomeric envelope glycoprotein from one of these X4 tropic viruses, MVP15132, bound HIV-1 CD4i monoclonal and polyclonal antibodies in a CD4-induced manner just as efficiently as did 7312A (
The discovery that sCD4-triggered HIV-2 is susceptible to binding and neutralization by HIV-1 elicited CD4i antibodies has practical application in studies of HIV-1 natural history and vaccine assessment. A number of investigative groups have attempted to stabilize the HIV-1 envelope glycoprotein in a CD4-bound configuration in order to use it as an immunogen designed to elicit antibodies against viral receptor surfaces or other intermediate envelope structures (Xiang et al. (2002) J Virol 76:9888-9899; Liao et al. (2004) J Virol 78:5270-5278; Fouts et al. (2000) J Virol 74:11427-11436). But methods to selectively identify and titer Nabs specific for such epitopes have been limited. Here, we show that neutralization of sCD4 treated HIV-2 represents an extremely sensitive and specific assay to detect HIV-1 elicited CD4i antibodies. Investigators have also targeted the membrane-proximal external region (MPER) of HIV-1 gp41 for vaccine development, since conserved epitopes in this region are capable of eliciting broadly reactive Nabs in natural infection (Purtscher et al. (1994) AIDS Res Hum Retroviruses 10:1651-1658; Buchacher et al. (1994) AIDS Res Hum Retroviruses 10:359-369; Zwick et al. (2001) J Virol 75:10892-10905; Ho et al. (2002) Vaccine 20:1169-1180; Liang et al. (1999) Vaccine 17:2862-2872; McGaughey et al. (2003) Biochemistry 42:3214-3223; Tian et al. (2002) J Pept Res 59:264-276; Barnett et al. (2001) J Virol 75:5526-5540; Mascola et al. (1996) J Infect Dis 173:340-348; Binley et al. (2004) J Virol 78:13232-13252; Ofek et al. (2004) J Virol 78:10724-10737). But again, neutralization assays are lacking that allow for the sensitive and specific detection of MPER epitope-specific Nabs (Opalka et al. (2004) J Immunol Methods 287:49-65). We thus considered the possibility that HIV-2 could act more generally as a “molecular scaffold” on which to present these and other HIV-1 epitope-specific antigens in the context of a functional envelope glycoprotein that does not otherwise cross-react with HIV-1 neutralizing antibodies. In recent studies, we have identified and modified by site-directed mutagenesis HIV-2 strains that can be used to detect and quantify binding and neutralization by the HIV-1 gp41 MPER-elicited human monoclonal antibody 4E10 with high sensitivity and specificity (F.B.R., J.M.D. and G.M.S., unpublished data). Thus, the strategy described in this report of using HIV-2 envelope glycoproteins in the context of infectious virions or as isolated proteins to detect HIV-1 epitope-specific antibodies may find wider application in the assessment of candidate vaccines and in studies of HIV-1 natural history.
With many HIV-1 vaccine candidates currently in the research pipeline, methods are needed for detecting and quantifying epitope-specific neutralizing antibody (Nab) responses in naturally-infected individuals and vaccinated subjects. HIV-1 and HIV-2 share less than 50% sequence similarity in envelope and they generally exhibit little cross-neutralization. We postulated that HIV-1 Nab epitopes could be identified in, or molecularly engineered into, functional HIV-2 env glycoproteins.
Sequence alignments of HIV-1 and HIV-2 viruses were examined to identify conserved regions in the membrane proximal external region (MPER) of gp41 and site-directed mutagenesis was used to change selected amino acids in this region of HIV-2 to resemble HIV-1. HIV-2 virions bearing envelopes with 4E10 core epitope amino acids, or control viruses containing wild-type HIV-1 or HIV-2 env, were analyzed for neutralization susceptibility to a panel of HIV-1 and HIV-2 monoclonal antibodies (Mab) or HIV-1 infected patient plasma using a JC53b1-13 HIV entry assay previously described (Nature 422:307, 2003).
The neutralization of HIV-2 by 4E10 and 2F5 monoclonal antibody was demonstrated. HIV-2 viruses 7312A, UC1, and ST were pre-incubated for 1 hour at 37° C. with the indicated concentrations of 4E10 and 2F5 monoclonal antibody. They were then plated on JC53b1-13 cells and infectivity determined after 48 hrs, as described in Decker et al (submitted and incorporated into this patent application). Site-directed mutations in the HIV-2 7312A envelope at positions 675 (L to I) and 676 (A to T) making the sequence of the 4E10 epitope identical to that of HIV-1 YU2 (see inset of
More specifically, virus bearing a prototypic HIV-1 env glycoprotein (YU2) was intermediately sensitive to neutralization by 4E10 (IC50=25 ug/ml), 2F5 (IC50=25 ug/ml), and b12 (IC50=3 ug/ml). Virus containing the envelope of HIV-2 strain 7312A was resistant to neutralization by all three Mabs (IC50>50 ug/ml). Site-directed substitution of aa 675 (L to I) and aa 676 (A to T) in the 7312A MPER (HXB numbering) rendered the virus remarkably sensitive to neutralization by 4E10 (IC50=0.8 ug/ml) (See,
In a similar fashion, site-directed mutations in the HIV-2 7312A envelope at positions 660 (K to A), 662 (N to D), 663 (S to K), and 665 (D to A), which together make the HIV-2 sequence identical to that of the 2F5 epitope region of HIV-1 YU2, rendered the modified HIV-2 virus susceptible to 2F5 with an IC50 of <0.1 ug/ml; conversely, the wild-type HIV-2 7312A envelope-containing viruses were completely resistant to 2F5 (IC50>50.0 ug/ml) (data not shown). These data show that certain naturally-occurring or genetically-modified strains of HIV-2 can be used to detect HIV neutralization by 4E10 and 4E10-like antibodies and by 2F5 and 2F5-like antibodies.
Conclusions: Naturally occurring or genetically engineered variants of HIV-2 env glycoprotein can be used to detect and quantify HIV-1 elicited 4E10-like and 2F5 Nabs with great sensitivity (IC50=0.1 ug/ml) and specificity. We have evidence that an analogous approach is feasible for detecting HIV-1 elicited Nabs against other MPER epitopes as well as epitopes on gp120. Epitope-specific assays of HIV-1 Nab responses may play an important role in HIV vaccine development and clinical assessment.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
The research underlying a portion of this invention was supported in part with funds from National Institute of Health grant no. U01 AI41530. The United States Government may have an interest in the subject matter of this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/11928 | 4/8/2005 | WO | 10/19/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60562824 | Apr 2004 | US | |
| 60606053 | Aug 2004 | US | |
| 60649551 | Feb 2005 | US |
