WO2003072045A2 - Treatment and prevention of aids progression and methods of using same - Google Patents

Treatment and prevention of aids progression and methods of using same Download PDF

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WO2003072045A2
WO2003072045A2 PCT/US2003/005514 US0305514W WO03072045A2 WO 2003072045 A2 WO2003072045 A2 WO 2003072045A2 US 0305514 W US0305514 W US 0305514W WO 03072045 A2 WO03072045 A2 WO 03072045A2
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ccr5
hiv
cxcr4
cells
protein
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WO2003072045A3 (en
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Ghalib Alkhatib
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Indiana University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors

Definitions

  • the present invention relates to methods and compounds for treating HIV. More specifically, the present invention relates to methods of preventing the transmission and progression of AIDS.
  • HIV-1 is the causative agent of acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • the virus specifically targets helper T lymphocytes and cells of the monocyte- macrophage lineage through high affinity interaction with the CD4 surface antigen (reviewed in Berger, et al., Broder, et al.).
  • CD4 surface antigen
  • a major discovery in 1996 concluded that the second receptor (coreceptor) used by HIV-1 belonged to a large family of seven transmembrane G- protein coupled receptors (Feng, et al.).
  • the CXC chemokine receptor CXCR4 is the coreceptor used by X4 HIV-1 isolates (Feng, et al.) while the CC chemokine receptor CCR5 serves as the coreceptor for R5 HIV-1 strains (Doranz, et al., Choe, et al., Alkhatib, et al., Deng, et al., Dragic, et al.).
  • the membrane orientation model of the 7TM domains chemokine receptor proteins (CCR5 is an example) is known to those in the art.
  • the amino acid sequence of the translated protein predicts seven transmembrane domains, an extracellular amino terminus and three extracellular loops, an intracellular carboxy terminus, three intracellular loops, and a S-S-proposed disulfide bond.
  • the conserved serine/Threonine rich cytoplasmic tail is the proposed domain involved in G-protein-coupled signaling.
  • Chemokine receptors share an overall 30% amino acid homology. Members of the same family share a higher degree of homology.
  • CXCR4 and CCR5 the major coreceptors for TCL-tropic and M-tropic HIV-1 , and other chemokine receptors such as CCR2b (Doranz, et al.), CCR3 (Doranz, et al., Alkhatib, et al., Choe, et al.), CCR8 (Rucker, et al.), STRL33 (BONZO) (Liao, et al., Deng, et al.) BOB (GPR15) (Deng, et al.) were also found to serve as fusion cofactors for HIV-1 entry. Despite the large number of related molecules showing an ability to employ alternate coreceptors in entry and infection, the principal coreceptors remain the initially discovered CXCR4 and CCR5 molecules.
  • HIV-1 binds to CD4 via an interaction between the first domain of CD4 and a discontinuous region of the external subunit of HIV-1 envelope glycoprotein, gp120. This region is referred to as the CD4 binding site (reviewed in Moore, et al.).
  • An accepted model for viral entry states that gp120 subunit binds first to CD4 resulting in a conformational change followed by interaction with the coreceptor which leads into another conformational change that exposes the fusion peptide of gp41 initiating the process of membrane fusion (reviewed in Moore, et al.).
  • CD4-CCR5 interaction demonstrated that the concentrations of CD4 and CCR5 required for efficient R5 infection are interdependent and that the requirements for each are increased when the other component is present in a limiting amount (Platt, et al.).
  • R5 infection requires the concerted actions of multiple CCR5 molecules and provided a mathematical model where 4-6 CCR5 monomers can be involved during R5 entry (Kuhmann, et al.).
  • CXCR4 can have the same requirement to form a complex that is essential for X4 infection (Dimitrov, et al.). Although these studies suggest physical interaction between CD4- coreceptor, the optimal stoichiometry of this association remains unknown.
  • Genotypes are represented by (+) for a wild-type allele and (-) for a ⁇ 32 allele.
  • ⁇ 32 homozygotes are referred to as -/-, ⁇ 32 heterozygotes as +/-, and to those with wild type CCR5 as +/+.
  • the amino acid structure of the ⁇ 32 mutant protein has been predicted based on the transmembrane structure of the wild type CCR5 sequence.
  • the ⁇ 32 deletion is located in a region corresponding to the second extracellular loop and results in a frameshift that produces a smaller protein which lacks the last three transmembrane domains and the carboxy terminal tail involved in G-protein signaling.
  • the frameshift caused by ⁇ 32 deletion introduces 31 new amino acid residues that are not encoded by CCR5.
  • the importance of chemokine receptors in HIV-1 transmission is highlighted by the finding that individuals homozygous for a 32-base pair deletion in CCR5 ( ⁇ 32/ ⁇ 32) are resistant to HIV-1 infection.
  • the deletion resulted in a frameshift mutation that introduced 31 new amino acid residues at the carboxy terminus of ⁇ 32 that are not present in CCR5.
  • the defective coreceptor gene encodes a prematurely terminated protein that is not detected at the cell surface and therefore is not functional as a fusion coreceptor (Samson, et al., Dean, et al., Huang, et al., Zimmerman, et al., Liu, et al.).
  • Genotypic analysis of this mutation and its distribution revealed that ⁇ 32 has a high allele frequency among Caucasians but was absent in African or Asian populations (Samson, et al., Dean, et al., Zimmerman, et al., Liu, et al.).
  • the mutant allele is not associated with any obvious phenotype in uninfected homozygous individuals.
  • Heterozygotes CCR5/ ⁇ 32 are not protected against infection, but once they become infected, have a slower progression to AIDS (Samson, et al., Dean, et al., Zimmerman, et al., Liu, et al.), indicating that partial resistance can occur in the presence of a single copy of the mutant CCR5 gene.
  • ⁇ 32 protein expression was analyzed in two brothers that are homozygous for the ⁇ 32 allele; one infected and the other uninfected. The results confirmed that the protected brother expressed ⁇ 32 protein whereas the infected brother lacked such expression. The absence of ⁇ 32 protein expression in this infected homozygote implicates a critical role for the protein in resistance to HIV-1. A growing body of evidence suggests that ⁇ 32 heterozygosity is associated with reduced risk to some complications of the disease. For example, studies analyzing the effect of +/- genotypes indicated reduced prevalence of ⁇ 32 mutation in those who do develop AIDS dementia complex (ADC) (van Rij, et al.).
  • ADC AIDS dementia complex
  • HAART highly active antiretroviral therapy
  • the resistance problem is particularly challenging because of the extraordinarily high HIV-1 mutation rate, and the ability of viral variants harboring resistance mutations in both reverse transcriptase and protease to continue replicating in vivo.
  • the viral mutability provides a rationale for developing alternate treatments.
  • the cellular receptors involved in HIV-1 entry are receiving special attention, with numerous candidate inhibitors at various stages of clinical development (Eckert, D. M. &Kim, P.S. (2001) Annu. Rev. Biochem. 70:777- 810).
  • the ⁇ 32 approach can therefore be useful to develop new methods and treatments of HIV since the ⁇ 32 protein is naturally occurring and is expressed in people who resist HIV infection.
  • Such a ⁇ 32-based treatment does not have the side effects found in the art HAART treatment of HIV-1 infection since it is aimed at decreasing the co-receptor density in a manner that is targeting both CCR5 and CXCR4, the major molecules that are responsible for disease transmission and progression, respectively.
  • individuals expressing the ⁇ 32 protein are healthy and do not show any immunological disorders.
  • a method of protecting individuals from contracting HIV by administering either a vector or a vaccine containing a sequence encoding the ⁇ 32 mutant protein containing the 31 frame shift amino acids. Also provided is a method of decreasing the amount of HIV coreceptors present on the cell surface by administering a compound having a sequence encoding the ⁇ 32 mutation in a pharmaceutically acceptable carrier. Also provided is a compound for decreasing the amount of HIV co-receptors present in the cell surface, the compound having a sequence encoding the ⁇ 32 mutant protein including the frame shift amino acids in a pharmaceutically acceptable carrier. A vector containing a sequence encoding the ⁇ 32 mutant protein is also provided. An assay for testing the efficacy of HIV treatment is also provided, the assay includes a detector for detecting the presence of the ⁇ 32 mutant protein in cells.
  • Figure 1 is a diagram showing the membrane orientation model of the 7 TM domain chemokine receptor protein
  • Figure 2 is a model showing the predicted amino acid structure of the ⁇ 32 mutant protein
  • Figures 3 A and B are graphs showing the fusion specificities of human cells expressing an adenovirus encoded CCR5 ( Figure 3A) or a vaccine virus encoded CXCR4 ( Figure 3B);
  • Figures 4A-C are flow cytometry analyses of cell surface CCR5 in a presence or absence of ⁇ 32;
  • FIGS 5 A and B are graphs showing the specific down modulation of CXCR4
  • Figures 6 A and B are graphs showing that the cells co-expressing ⁇ 32 and either CCR5 or CXCR4 are resistant to R5 and X4 fusion;
  • Figures 7 A and B are graphs showing the specificity of ⁇ 32 induced inhibition of HIV-1 Env-mediated cell fusion;
  • Figures 8 A and B are graphs showing the cell specificity of the ⁇ 32 affect;
  • Figures 9 A and B show PHA plus IL-2 activation upregulates CXCR4 and CCR5;
  • Figures 10 A and B are histograms representing staining of uninfected PMBC cells;
  • Figures 1 1 A and B are graphs showing the affect of ⁇ 32 on Env-mediated cell fusion and human PMBCs;
  • Figures 12 A-C are graphs showing PMBCs from individuals with known CCR5 genotype
  • Figures 13 A and B are graphs showing the infection kinetics of -/- and +/+ PMBCs with HIV-1 IIIB(X4) and Ba-L(R5);
  • Figures 14 A-C are photographs showing immunoblot analysis of ⁇ 32 and CCR5 proteins expressed in infected 293 cells;
  • Figures 15 A and B are photographs showing the immunodetection of native ⁇ 32 protein expressed in unstimulated PMBCs of three -/- homozygous individuals;
  • Figures 16 A-C are photographs showing the expression analysis of ⁇ 32 mRNA in -/- PMBCs ( Figure 16A) and recombinant 85 infected cells ( Figure 16B) by RT-PCR;
  • Figure 17 is a graph showing the effect of recombinant ⁇ 32 protein in X4 infection
  • Figures 18 A and B are graphs showing the Phytohemagglutinin-A + IL2- activated Ficoll purified human PBMCs which were infected with either vLA-1 (Ad5/CCR5) or vLA-2(Ad5/ ⁇ 32) at 3pfu per cell for each virus for two days that were then infected with either Ba-L(R5) or IIIB(X4);
  • Figures 19 A and B are hypothetical models of the ⁇ 32 affect; and DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides a method and composition for treating and vaccinating against HIV.
  • the composition of the present invention can be used either as a treatment for an individual who has already contracted HIV or as a vaccine to prevent the transmission of HIV. Accordingly, the composition must be administered for a sufficient period of time or at a sufficient concentration to obtain the desired effect in the individual to whom the composition has been administered.
  • composition of the present invention includes a sequence encoding the ⁇ 32 mutant protein and analogues and homologues thereof and a pharmaceutically acceptable carrier.
  • the composition can be used to create a vaccine or as a gene therapy.
  • composition of the present invention can be administered in any manner known to those of skill in the art.
  • the composition is administered either orally or intramuscularly.
  • An adenovirus vector encoding the ⁇ 32 mutant protein can be used as a vaccine which is preferably administered orally.
  • An acceptable vaccine that uses the same vector used to vaccinate against smallpox can be used to vaccinate individuals.
  • vaccine as used herein, the term is intended to include, but is not limited to a treatment which prevents HIV infection in individuals who have received the treatment thereby making the individual immune against HIV.
  • a recombinant vaccinia virus encoding the ⁇ 32 mutant protein has been constructed and used to infect human peripheral blood lymphocytes. Lymphocytes expressing the encoded ⁇ 32 mutant protein showed resistance to HIV-1 entry and infection.
  • a later administration of the vaccine can be given every month to maintain sufficient expression of the encoded ⁇ 32 mutant protein.
  • the clinical condition of the individual patient, the site and method of administration, scheduling of administration, and other factors known to medical practitioners can be taken into consideration.
  • the "effective amount" for purposes herein is thus determined by such considerations as are known in the art of vaccination wherein it must be effective to provide measurable anti-virus titer in persons given the vaccine, and, in a preferred embodiment, persons who are non-responsive to a standard anti-retroviral therapy.
  • titers can be determined, as well as proliferative assays in response to viral antigen can be run as are well known in the art.
  • the desired effect of the treatment is to either prevent the transmission of HIV or to prevent the progression of HIV infection in seropositive individuals.
  • the composition accomplishes these effects by preventing effectively the expression of coreceptors (CCR5 and CXCR4) present on the cell surface responsible for transmission and progression.
  • gene therapy refers to the transfer of genetic material (e.g DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype.
  • the genetic material of interest encodes a product (e.g. the ⁇ 32 mutant protein) whose production in vivo is desired.
  • the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value.
  • the genetic material of interest encodes a suicide gene.
  • ex vivo and (2) in vivo gene therapy Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy.
  • ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro.
  • a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient.
  • These genetically reimplanted cells have been shown to express the transfected genetic material in situ.
  • target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ that is within the recipient.
  • the gene is repaired in situ (Culver, 1998). These genetically altered cells have been shown to express the transfected genetic material in situ.
  • the gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell.
  • the expression vehicle can include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR of the gene can be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore as used herein the expression vehicle can, as needed, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred and only include the specific amino acid coding region.
  • the expression vehicle can include a promotor for controlling transcription of the heterologous material and can be either a constitutive or inducible promotor to allow selective transcription.
  • Enhancers that can be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non- translated DNA sequence, which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter.
  • the expression vehicle can also include a selection gene as described herein below.
  • Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Ml (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Ml (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see United States patent 4,866,042 for vectors involving the central nervous system and also United States patents 5,464,764 and 5,487,992 for positive
  • nucleic acids by infection offers several advantages over the other listed methods. Higher efficiency can be obtained due to their infectious nature.
  • viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.
  • DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector Adenop53TK.
  • This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences.
  • TK herpes virus thymidine kinase
  • This vector can be used to infect cells that have an adenovirus receptor, which includes most cancers of epithelial origin as well as others.
  • This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vitro or ex vivo culture of cells, a tissue or a human subject.
  • Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy.
  • Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus.
  • An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation does not occur.
  • Such features include, for example, promoter and regulatory elements that are ⁇ ⁇ specific for the desired -cell type.
  • recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity.
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms.
  • viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • the vector to be used in the methods of the invention depends on desired cell type to be targeted and is known to those skilled in the art. For example, if breast cancer is to be treated then a vector specific for such epithelial cells can be used.
  • a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell can be used.
  • a recombinant adeno-associated (AAV) viral vector encoding the ⁇ 32 mutant protein can be constructed and used to infect hematopoietic stem cells.
  • AAV vectors are potential gene delivery viral vectors that have the advantage of being non-pathogenic to human cells. Such vectors can provide a safe delivery and integration of the ⁇ 32 gene into hematopoietic progenitor cells. When transplanted into the patient, these cells can give rise into blood lymphocytes that express the protective ⁇ 32 mutant protein.
  • the cells expressing the ⁇ 32 mutant protein are immune to infection by a wide range of HIV-1 strains including X4, R5, and X4R5.
  • Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection.
  • the genome of the virus is modified so that it maintains all the necessary genes, regulatory ' I sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles, which are capable of undergoing further rounds of infection.
  • the vector's genome is also engineered to encode and express the desired recombinant gene.
  • the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed do not contain a genome and therefore cannot proceed through subsequent rounds of infection.
  • the specific type of vector depends upon the intended application.
  • the actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.
  • the recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neurodegenerative diseases. Following injection, the viral vectors circulate until they recognize host cells with the appropriate target specificity for infection.
  • An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid.
  • Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal.
  • non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like.
  • Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art.
  • the compound of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
  • the doses can be single doses or multiple doses over a period of several days, but single doses are preferred.
  • the doses can be single doses or multiple doses over a period of several days.
  • the treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.
  • the ⁇ 32 mutation is also an effective therapeutic target for the treatment of HIV-1 infection.
  • This target can be used for developing further compositions for treating HIV infection.
  • new compounds can be developed which target both CCR5 and CXCR4 coreceptors and thereby inhibiting HIV transmission and progression.
  • additional gene therapy products such as new vectors can be developed which target the major HIV coreceptors.
  • Studies analyzing the molecular effect of this mutation have largely relied on the introduction of the ⁇ 32 DNA into cells by transfection. This method results in an inefficient delivery of the ⁇ 32 into the cells and results in expression of ⁇ 32 protein in very few cells in a sample monolayer.
  • Applicants have developed adenovirus and vaccinia virus vectors that deliver the ⁇ 32 DNA efficiently into every cell in the monolayer. As a result, 100% of the cells transduced with the ⁇ 32 vector express this protein ( ⁇ 32 protein).
  • ⁇ 32 heterozygosity (individuals carrying one copy of ⁇ 32) is associated with reduced risk to some complications of the disease.
  • studies analyzing the effect of ⁇ 32 genotypes indicated reduced prevalence of ⁇ 32 mutation in those who do develop AIDS dementia complex (ADC).
  • Others reported an association of ⁇ 32 mutation with protection against HIV-associated lymphoma.
  • Recent reports described that carriers of the ⁇ 32 mutation respond better to highly active antiretroviral therapy (HAART) treatment compared to wild type controls.
  • HAART highly active antiretroviral therapy
  • CD8+ T cells purified from ⁇ 32 carriers showed increased ability to kill virally infected cells. All the above studies point to the potential use of the ⁇ 32 effect in other disease conditions.
  • HIV-1 Human immunodeficiency virus type 1
  • CD4 a coreceptor, principally the CCR5 and/or CXCR4 chemokine receptors, for entry into host cells.
  • CCR5 The central role of CCR5 in HIV-1 transmission and pathogenesis has been highlighted by the epidemiological and genetic identification of powerful disease modifying effects of the naturally occurring CCR ⁇ 32 allele, a 32 base pair deletion encoding a truncated and non-cell surface expressed version of the receptor.
  • CCR ⁇ 32 homozygotes are rarely found among HIV-1 infected individuals.
  • HIV-1 infected CCR ⁇ 32 heterozygotes progress more slowly to AIDS than individuals lacking this allele.
  • Previous studies have indicated that ⁇ 32 protein binds to wild-type CCR5 inside the cell and can retard the transport of functional CCR5 to the cell surface.
  • this mechanism did not explain why CXCR4 only rarely could compensate for the CCR5 deficiency and allow X4 or dual tropic (R5X4) virus infection.
  • the present invention shows that the ⁇ 32 protein down-regulates the major coreceptors resulting in an unfavorable of the molecules involved in membrane fusion.
  • PBMCs from ⁇ 32/ ⁇ 32 individuals express lower surface levels of CXCR4 in comparison to wild-type CCR5 PBMCs. Further, lower X4 fusion/infection activity was correlated in the ⁇ 32/ ⁇ 32 cells.
  • replication- defective adenovirus type 5 (Ad5) vectors were constructed encoding either CCR5 or ⁇ 32 proteins.
  • the Ad5 gene delivery system results in efficient delivery of the gene of interest to most cells in a monolayer resulting in sufficient expression levels to allow detailed analysis and characterization of the ⁇ 32 protein and examination of its potential role in the protective phenotype.
  • One mechanism of genetic resistance to HIV-1 is caused by the unique activity of the ⁇ 32 protein that results in an unfavorable stoichiometry of the surface proteins involved in HIV-1 entry.
  • ⁇ 32 interacts specifically with the major coreceptors CXCR4 and CCR5 and impairs the formation of coreceptor complexes and CD4-coreceptor interaction resulting in an imbalance of the optimal stoichiometry of the molecules involved in the process of membrane fusion.
  • the experimental approach described herein enables one to characterize the ⁇ 32 protein at the molecular level and determine its contribution to resistance to HIV-1 infection.
  • the observed effect of ⁇ 32 on both CCR5 and CXCR4 implicates a common interaction site for the principal coreceptors on ⁇ 32.
  • ⁇ 32 protein encoded by a recombinant vaccinia virus was also studied. This can also be in the form of an attenuated vaccinia virus that is used for smallpox vaccination.
  • a recombinant vaccinia virus that expresses the ⁇ -32 protein with expression that results in cells that are resistant to HIV-1 entry has also been established. The results of the experiment are shown in Figure XY.
  • Cells (MAGI-CCR5) were infected with recombinant vaccinia viruses encoding the proteins indicated at the X-axis. These cells were challenged with cells expressing the two major prototypic HIV-1 envelope glycoproteins (LAV or Ba-L).
  • the extent of cell fusion was determined by measuring the production of ⁇ - galactosidase. The results show that cells expressing ⁇ 32 are resistant to HIV-1 fusion.
  • Two controls were included in this experiment. The first control is the use of cells infected with the vaccinia vector (WR), the vector used to generate the ⁇ 32/vaccinia recombinant virus. The second control is the use of cells infected with a vaccinia recombinant encoding CCR5. The two controls showed no resistance to HIV-1 fusion as demonstrated in the figure.
  • ⁇ 32 protein was also investigated. Delta-32 RNA and protein expression in (-/-) PBMCs was also examined. RNA expression of ⁇ 32 was verified by RT-PCR analysis. The immunoblot analysis revealed a protein band that corresponds to the same molecular weight band seen with recombinant protein analysis. These protein bands were not obtained with the preimmune serum. The identity of the band that appears above the 34 Kd marker band is not known at present, however, since it is expressed in normal CCR5 individuals, it could represent a cross-reactive cellular protein. The analysis also revealed that the ⁇ 32 protein band detected in the protected (-/-) individuals was absent in an infected (-/-) individual suggesting a critical role for the ⁇ 32 protein in resistance to HIV-1.
  • the recombinant vaccinia virus encoding the ⁇ 32 protein can be used as a live attenuated vaccine to deliver the ⁇ 32 protein into susceptible cells.
  • the mechanism of protection obtained with this recombinant virus will be investigated in a mouse model that expresses human CXCR4, CCR5 and CD4.
  • the mode of administration can be intramuscular or intravenous.
  • the C-terminal region of the ⁇ 32 protein created novel epitopes that are not native to people who do not express ⁇ 32 protein. Therefore, antibody generation to those novel epitopes in ⁇ 32 occur in vaccinated mice. These antibodies can be examined for their ability to neutralize HIV-1 infection.
  • Adeno-associated viral vectors are also potential vectors for gene therapy (Zhou, et al., Gene Therapy, 3:223-229 (1996)). These vectors also contain the herpes virus TK promoter-driven neomycin gene for selection. AAV- based vectors have the advantage of being non-pathogenic and the fact that they integrate into the host chromosome by a site-specific manner. Therefore, they do not appear to lead to insertional mutations. HIV-based lentivirus vectors can also be utilized. Recombinant HIV-1 vector expressing the ⁇ 32 protein can be constructed and used to treat HIV-1 infection in cultured peripheral blood lymphocytes that are chronically infected with HIV-1.
  • the idea is that the recombinant ⁇ 32/HIV integrates into the host chromosome at the same sites that contain the wild type genomic copies of HIV-1. Recombination results in the insertion of the ⁇ 32 gene into the host chromosome thereby providing continuous expression of the ⁇ 32 protein.
  • This approach can be very beneficial for those patients who do not respond to HAART and are therefore left to their fate and to the virus controlling their immune systems.
  • the idea of generating HIV-based vectors to trap HIV-1 virus has been previously suggested (Endres, et al., Science, 278:1462-1464 1997). This approach offers a method for delivering the ⁇ 32 protein directly to HIV-infected cells in vivo and provides an additional treatment strategy in conjunction with existing HAART therapy.
  • PCR Polymerase chain reaction
  • Example 1 In order to study the role of ⁇ 32 in genetic resistance to HIV-1 infection, a vector system is needed that can reproducibly deliver the ⁇ 32 transcription unit into most cells in a monolayer that result in sufficient quantities of recombinant ⁇ 32 in a variety of cell types.
  • the immunological reagents must also be developed that enable one to specifically detect native and recombinant ⁇ 32 proteins.
  • a number of cDNA clones have been obtained encoding several CC and CXC chemokine receptors. Most of these receptors were subcloned under the control of the synthetic early/late promoter of vaccinia virus to produce higher levels of expression of the chemokine receptor protein.
  • the original cDNA clones were all under the T7 promoter, and are used when lower levels expression of the protein is desired.
  • a number of recombinant vaccinia viruses made by Chris Broder were obtained from the laboratory of Ed Berger, N1AID.NIH. These recombinants are used to prepare cells expressing a variety of HIV-1 envelope glycoproteins (Envs) derived from different X4, R5, or R5/X4 isolates.
  • Envs HIV-1 envelope glycoproteins
  • the immuno-detection of recombinant proteins is critical to verify expression, localization, and quantification of the molecules being tested.
  • Polyclonal antibodies against synthetic peptides corresponding to the amino termini of CXCR4 (Feng, et al.) and CCR5 (Alkhatib, et al.) have been described and are available for use in the laboratory. It was shown that these antibodies detect recombinant CXCR4 expressed by using the vaccinia virus vectors, and endogenous CXCR4 made in peripheral blood macrophages and lymphocytes.
  • the anti-CXCR4 peptide antibodies do not detect surface CXCR4.
  • monoclonal antibodies are used that have been obtained commercially.
  • ⁇ 32 a recombinant adenovirus encoding either wild type CCR5 or the deletion mutant ⁇ 32 protein, referred to as ⁇ 32 has been constructed.
  • the entire cDNA fragments encoding either ⁇ 32 or CCR5 were individually
  • Ad5/CCR5 DNA was used to isolate positive recombinant viral plaques on 293 cell monolayers. Southern blot analysis was subsequently performed to confirm the predicted structure of recombinant viruses without deletions or rearrangements, even after three rounds of plaque purification and propagation. The recombinant viruses consistently grew to titers ranging between 7x10 8 to 1x10 9 plaque forming units (PFU)/ml in 293 cells.
  • PFU plaque forming units
  • Ad5/MVHA Ad5/MVHA
  • Ad ⁇ MVPC Ad5/MVHA
  • Ad ⁇ MVPC Ad5/MVHA
  • Ad ⁇ MVPC recombinant Ad5 viruses encoding either measles virus (MV) haemaglutinin (Alkhatib, et al.) or MV phosphoprotein Alkhatib, et al.) were used as negative controls for all subsequent experiments to measure the effect of recombinant ⁇ 32 protein on cell surface expression of the major coreceptors and their fusion activities.
  • Wild type Ad5 is a replication competent virus that does not contain DNA sequences that are specific for ⁇ 32 or any chemokine receptor. Expression of MVHA or MVPC by Ad5 was verified by surface staining using MV-specific polyclonal antibodies as previously described (Alkhatib, et al.).
  • the experimental approach involves the analysis of fusion between two distinct cell populations, one expressing CD4 (endogenous or encoded by a recombinant virus) and the other expressing the indicated HIV-1 envelope glycoprotein encoded by a recombinant vaccinia virus.
  • Cell fusion is scored by a reporter gene activation assay in which the cytoplasm of one cell population expressing vaccinia virus-encoded T7 RNA polymerase and the cytoplasm of the other expressing lac Z gene linked to the T7 promoter; cell fusion activates ⁇ - Galactosidase.
  • the advantages of preparing target cells (cells expressing the coreceptor) using the Ad5 expression system include:
  • the Ad5 cytopathic effect is much less severe than vaccinia, 2) The cells are healthier and easier to manipulate; 3) The recombinant Ad5 viruses are efficient high-level expressing vectors that result in infection of all cells in a monolayer; 4) The G-protein signaling pathway is not impaired in Ad5-infected cells (Braciak, et al.) compared to vaccinia virus infected cells; 5) The recombinant Ad5 viruses are replication-defective in cells other than 293 and can be useful to analyze without the cytopathic effect associated with the wild type virus; and 6) The recombinant Ad5 viruses provide a much better signal to background ratio.
  • Co-expression of proteins is accomplished with the Ad5 vector system by infecting cells with two different recombinant viruses at the same time. This is a well established method for expressing two different proteins in a variety of cell types due to the broad host specificity of Ad5 for mammalian cells (Alkhatib, et al.).
  • the basic features of the fusion assay were developed by using the HIV-1 Env-CD4 interaction of two different populations of cells, one expressing CD4 and the other expressing the HIV-1 Env (Nussbaum, et al., Alkhatib, et al.).
  • adenovirus expression system The rationale for using an adenovirus expression system is that cells infected with this vector can, unlike vaccinia infected cells, respond to calcium flux assays making it possible to analyze the response of infected cells to different chemokines. Moreover, the recombinant Ad5 viruses used are replication-defective and do not cause severe cytopathic effect in a wide range of mammalian cells including primary cells. In the vaccinia-based cell fusion assay, CCR5 functions as a fusion coreceptor for Envs from several R5-tropic HIV-1 isolates, as well as the R5/X4 strain 89.6 (Alkhatib, et al.).
  • human 293 cells were used as the target cell population after transfection with pCDNA3/CD4 and coinfection with Ad5T7 ⁇ -gal (lacZ under T7, from Frank Graham and vLA-1 (CCR5). Hela cells coinfected with vTF7-3 (T7 RNA polymerase) and one of the HIV-1 Envs as effector cells were used. After mixing the effector and target cell populations and incubation at 37°C for 2.5 hours, fusion specificity of HIV-1 Envs was measured by ⁇ -gal production in a colorimetric lysate assay.
  • Monoclonal antibodies to CCR5 were used to detect coreceptor density at the cell surface of infected cells. Surface levels of CCR5 were significantly reduced in the presence of recombinant ⁇ 32 protein ( Figure 4), consistent with previous published observations that examined the effect of ⁇ 32 on cell surface expression of CCR5 (Benkirane, et al., Wu, et al., Paxton, et al.).
  • a human CCR5/293 cell line and another CXCR2/293 cell line were used to analyze the effect of ⁇ 32 on CXCR4.
  • Human CCR5/293 cells were infected with VLA-2 ⁇ 32), vLA-1 (CCR5), Ad ⁇ MVHA (MVHA), Ad ⁇ MVPC (MVPC), or Ad5 and stained with Mabs against CXCR4.
  • the results of this analysis demonstrate that cells expressing ⁇ 32 but not CCR5, MVHA, or MVPC showed downmodulation of CXCR4 ( Figure 5A).
  • CXCR2 is a CXC chemokine receptor that shares 35% homology with CXCR4.
  • CXCR2 or CXCR4 surface levels were not significantly altered in cells expressing either CCR5 or MVHA - ( Figure 6B, CCR5/CXCR2 and MVHA/CXCR4 line graphs) indicating the specificity of ⁇ 32 downmodulation effect.
  • EGFR epidermal growth factor receptor
  • a glycoprotein expressed on most cells including 293 cells and on CCR2 in a human CCR2/293 cell line were examined.
  • FIG. 4 shows the flow cytometry analysis of cell surface CCR5 in the presence or absence of ⁇ 32.
  • Cells (293 cells) expressing CCR5 and/or ⁇ 32 were treated with monoclonal antibodies and detected by indirect staining using fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G. The values shown at the right corner of each histogram represent mean fluorescence intensity.
  • Figure 5 shows the specific downmodulation of CXCR4.
  • Figure 5A shows human 293 cells which were infected at 10 pfu/cell with the different Ad5 viruses encoding wild type adenovirus proteins (Ad5), measles virus hemaglutinin (MVHA), measles C& phosphoproteins (MVPC), CCR5, or ⁇ 32 and stained for surface CXCR4. Reduction in surface CXCR4 was only observed with cells expressing ⁇ 32 but not other proteins such as CCR5, MVHA, or MVPC.
  • Ad5 wild type adenovirus proteins
  • MVHA measles virus hemaglutinin
  • MVPC measles C& phosphoproteins
  • Figure 5B shows ⁇ 32-specific downmodulation of CXCR4 in a CXCR2/293 cell line.
  • Cells were infected at increasing moi's with either vLA-1 (CCR5/Ad5), Ad5/MVHA, or vLA-2 (Ad5/ ⁇ 32), then stained with monoclonal antibodies to CXCR4 or CXCR2 (R&D).
  • ⁇ 32 expression resulted in reduced surface staining of CXCR4 ( ⁇ /CXCR4) but had no significant effect on CXCR2 surface expression ( ⁇ /CXCR2).
  • Expression of MVHA, or CCR5 by the adenovirus system had no downmodulation effect on cell surface expression of CXCR4 or CXCR2.
  • a human 293/CD4 Cell line was generated and cells were infected with either vLA-1 (CCR5), vLA-2 ⁇ 32), Ad5, or coinfected with vLA-1+vLA-2 (CCR5+ ⁇ 32) then infected with Ad5Pol3 (T7 RNA polymerase).
  • vCB-21 R and either vCB-16 (Unc), . vCB-41 (LAV), or vCB-39 (ADA). Fusion activity was scored by the amount of ⁇ -galactosidase produced.
  • Figure 6 shows cells coexpressing ⁇ 32 and either CCR5 or CXCR4 are resistant to R5 and X4 fusion.
  • Human 293 cells expressing CCR5 and CXCR4 (endogenous), or coexpressing CCR5+ ⁇ 32 or ⁇ 32 were challenged with effector Hela cells expressing the indicated HIV-1 Envs. Cell fusion was scored by ⁇ -gal production. This analysis is representative of at least three different experiments.
  • X4 fusion was measured by a quantitative method while Lee, et al. used microscopic counting of syncytia.
  • HTLV-1 Env Recombinant vaccinia viruses encoding HTLV-1 Env or its transmembrane gp46 subunit were obtained from Dr. H. Shida. gp46 expression was used as a negative control for the specific HTLV-1 Env-mediated cell fusion. This choice was based on the fact that HTLV-1 is a human retrovirus that has a broad range specificity for mammalian cell lines (Sutton, et al.). Target cells expressing ⁇ 32 showed specific reduction in X4 fusion when challenged with the X4 LAV Env. In contrast, the same target cells challenged with HTLV-1 Env-expressing cells showed comparable levels of cell fusion observed with control cells expressing CCR5, MVHA or T7 RNA polymerase (pol3) ( Figure 7).
  • Figure 7 shows the specificity of ⁇ 32-induced inhibition of HIV-1 Env-mediated cell fusion.
  • Target cells (293 or MAGI- CCR5) were coinfected with Ad5pol3 (T7RNA polymerase) and either vLA-2 (pol3+ ⁇ 32) vLA 1 (pol3+CCR5).
  • Figure 8 shows results obtained by the quantitative assay of ⁇ -gal in detergent cell lysates.
  • X4 and R5 fusion showed dramatic reduction as a result of ⁇ 32 expression, whereas no significant effect on X4 fusion was observed with HL60.
  • Control cells expressing recombinant CCR5 did not show significant reduction in X4 fusion.
  • HL60 cells were induced to differentiate into macrophages by RA treatment, the differentiated cells showed the ⁇ 32 effect ( Figure 8B).
  • Figure 8 shows the cell specificity of the ⁇ 32 effect.
  • the effect of ⁇ 32 on X4 fusion is observed with MAGI-CCR5 and 293 cell lines but not with HL60 cell line Figure 8(A).
  • Retinoic acid (RA) treatment of HL60 cell induces them to differentiate and become sensitive to -the ⁇ 32 effect Figure 8(B).
  • Cells were transfected with pCDNA3/CD4 to provide CD4 expression. All cell types were coinfected with pol3 (no ⁇ 32) or vLA 2+pol3 (+ ⁇ 32) and challenged with Hela cells expressing X4 Env (LAV) or R5 (Ba-L) and pT7-lacZ. Cell fusion was measured by ⁇ -gal produced.
  • CD3+CD28 stimulation produces cells that are susceptible for X4 isolates but are resistant to R5 viruses (Carroll, et al.).
  • FIG. 9 shows that PHA+IL-2 activation upregulates CXCR4 and CCR5.
  • PBMCs from seven healthy individuals were stimulated with either PHA Figure 9(A) or PHA+IL-2 and used for cell surface staining of CCR5 and CXCR4 Figure 9(A) or cell fusion assay using one PBMC sample Figure 9(B).
  • CXCR4 levels are variable among individuals confirming published data (Lee, 1999).
  • unstimulated PBMCs respond well in signaling assays using SDF-1 for receptor activation. It appears that the levels of CXCR4 required for signaling assays are different from those required for fusion/infection.
  • the effect of recombinant ⁇ 32 was examined in PBMCs freshly isolated from healthy donors.
  • vLA-2 was used to express recombinant ⁇ 32 in PHA+IL-2 stimulated PBMCs and examined cell surface levels of CXCR4, CD4 and CD44 by FACS analysis. A slight downmodulation effect of CD4 (15-20%) and no effect on CD44 surface expression was observed ( Figure 10A). In contrast, appreciable downmodulation of CCR5 and CXCR4 was observed in the same PBMC population tested ( Figure 10B).
  • Ad5/CCR5 is a replication-defective recombinant virus that does not replicate in primary cells and therefore, it is not expected to result in a robust increase in the levels of CCR5.
  • CCR5 staining of Ad5/CCR5-infected PBMCs showed an increase in CCR5 expression (from 370 to 404, Figure 10B) confirming the delivery of recombinant CCR5 in primary cells.
  • PBMC sample was analyzed by infecting with vLA-1 , vLA2, or Ad5/MVHA and vCB- 21 R (T7-lacZ). Hela cells expressing T7 RNA polymerase and the X4 LAV or Unc Envs were used as effector cells. After mixing the two cell populations and allowing the fusion reaction to incubate for two hours, detergent-treated cell lysates were used to quantitate the amount of ⁇ -gal production as a result of cell fusion. The results demonstrate the ability of recombinant ⁇ 32 to inhibit X4 fusion in these primary cells ( Figure 11 A). PBMCs expressing recombinant CCR5 ( Figure 11 B) or MVHA showed no significant reduction in X4 fusion.
  • PBMC samples isolated from wild type +/+ and homozygous -/- individuals were used to compare cell surface expression of CXCR4.
  • PBMCs were stimulated with PHA+IL-2 for four days then used for FACS staining, cell fusion, and HIV-1 infection assays.
  • the results demonstrate a trend towards lower CXCR4 staining in -/- PBMCs ( Figure 12A).
  • Cell fusion activity with LAV (X4) of -/- cells is always lower th,an that observed with +/+ cells (Figure 12C).
  • Surface expression of CD4 was variable in both +/+ and -/- PBMCs and did not seem lower in -/- compared to +/+ cells ( Figure 12B). The observed CD4 variability is unlikely to influence the data presented here in terms of lower CXCR4 found in -/- individuals since it was observed in both +/+ and -/- samples.
  • Figure 1 1 shows the effect of ⁇ 32 on Env-mediated cell fusion in human PBMCs.
  • the same PBMC samples used for FACS staining in Figure 10B were used in this cell fusion analysis.
  • PBMCs were infected with either vLA-1 (Ad5/CCR5) or vLA-2 (Ad5/ ⁇ 32) at increasing PFUs/cell to ensure expressing recombinant protein in all cells in the monolayer.
  • Infected PBMCs were challenged with Hela cells expressing either Unc or the X4 LAV.
  • PBMCs expressing recombinant CCR5 Figure 1 1 (B) or MVHA Unlike PBMCs expressing recombinant CCR5 Figure 1 1 (B) or MVHA, PBMCs expressing recombinant ⁇ 32 showed a dramatic reduction in X4 fusion Figure 1 1 (A).
  • PBMCs were infected (10 5 cells) with the X4 lab adapted isolate 1MB or the R5 Ba-L and sample supernatants were collected every three days over a 15 days period.
  • the results of this experiment demonstrate that +/+ cells infected with IIIB produced higher amounts of p24 indicating a more efficient productive infection compared to -/- cells ( Figure 13).
  • CXCR4 expression in -/- homozygotes is different from that seen in normal +/+ individuals in terms of surface density and coreceptor function.
  • Figure 12 depicts the results of experiments where PBMCs from individuals with known CCR5 genotype were used. Genotypes are represented by (+) for a wild-type allele and (-) for a ⁇ 32 allele. PBMCs from -/- individuals express lower surface levels of CXCR4, show less cell fusion activity with X4 Env (LAV) and are less susceptible to X4 fusion than +/+ cells. PBMCs from -/- and +/+ individuals were stimulated with PHA+IL-2 for 4 days and used for staining Figure 12(A), cell fusion Figure 12(B), and IIIB infection.
  • LAV X4 Env
  • FIG. 13 shows the infection kinetics of -/- and +/+ PBMCs with HIV-1 IIIB (X4) and Ba-L (R5).
  • PBMCs were stimulated with PHA+IL-2 for 4 days and used in the infectivity assay. Infection was performed in a 96 well plate (10 5 cells/well).
  • Figure 14B shows the identity of the 34 KD band detected with the C-terminal antibodies.
  • the band above the 50 Kd marker band detected in both CCR5 and ⁇ 32 expressing cells could not be CCR5 since it migrates slower (above 50 Kd) than CCR5 that migrates around the 46 Kd marker band ( Figure 14 A).
  • Figure 14 shows the immunoblot analysis of ⁇ 32 and CCR5 proteins expressed in infected 293 cells. Cells were infected with vLA-1 , vLA2, or Ad5 at 10 pfu/cell for 16 hours.
  • FIG. 14(A) shows the monoclonal antisera detect CCR5 but not ⁇ 32 (10% SDS-PAGE);
  • Figure 14(B) shows the C-terminal antibodies detect ⁇ 32 (12% SDS-PAGE) ; and
  • Figure 14(C) shows N-terminal antibodies that detect both CCR5 and ⁇ 32 proteins (10.5% SDS PAGE).
  • ⁇ 32 protein expression in -/- PBMCs was examined using the ⁇ 32-specific antisera.
  • Cell lysates were prepared from unstimulated cells and analyzed the same way described for recombinant ⁇ 32 protein described above.
  • the immunoblot containing three -/- and three +/+ samples was probed with anti- ⁇ 32 antibodies generated against the carboxy terminus of ⁇ 32.
  • the analysis revealed a protein band of different intensity for each -/- individual that corresponds to the same molecular weight band seen with recombinant protein analysis (Figure 15).
  • Approximately, equivalent amounts of protein cell lysates were loaded in each lane. Equal gel loading was verified by staining a similar gel with commassie blue stain.
  • Figure 15 shows the immunodetection of native ⁇ 32 protein expressed in unstimulated PBMCs of three -/- homozygous individuals. The blot was probed with antibodies generated against the carboxy terminus of ⁇ 32 that specifically detect ⁇ 32 protein. Over-exposure did not show any ⁇ 32-related band in +/+ PBMCs. Samples (-/-) 1 , 2 and 3 on this blot correspond to #2, #3, and #6 respectively on Figure 12. Equivalent gel loading was verified by staining a similar gel with commassie blue stain.
  • Samples 1 and 2 in Figure 12(A) correspond to samples 1 &2 in Figure 16.
  • N represents a normal non-genotyped PBMC sample.
  • Southern blot analysis was performed on the gel shown in Figure 16(A) and probed with a 32 P-labeled CCR5/ ⁇ 32-specific DNA Figure 16(C).
  • CCR5 and ⁇ 32-related fragments could have been generated from smaller mRNA species.
  • vLA-1 CCR5
  • vLA-2 vLA-2 ⁇ 32
  • MAGI-CCR5 cells were infected with a recombinant Ad5 encoding T7 RNA polymerase (provided by Frank Graham, McMaster University) at 27 pfu/cell or with a recombinant adenovirus encoding MVHA (Alkhatib, et al.) at 27 pfu/cell and assayed at the same time for PHS 398.
  • Figure 17 shows the effect of recombinant ⁇ 32 protein on X4 infection.
  • MAGI-CCR5 cells were infected with vLA-1 (encoding CCR5) or vLA-2 (encoding ⁇ 32 protein) at increasing viral concentrations (PFU/cell) then infected with the X4 IIIB.
  • HIV-1 infection is quantitated by the amount of ⁇ -galactosidase produced as a result of HIV LTR activation that controls expression of ⁇ -galactosidase.
  • the infection protocol is adapted from Kimpton and Emerman. Values of ⁇ -gal production in cells infected with Ad5pol3 (encoding T7 RNA polymerase) or Ad5/MVHA at 27 pfu/cell were 68 and 60 respectively.
  • Ad5pol3 encoding T7 RNA polymerase
  • Ad5/MVHA at 27 pfu/cell
  • the ability of recombinant ⁇ 32 to provide protection against HIV-1 productive infection in vitro was examined.
  • the experiment was performed by using (vLA-2 ⁇ 32-encoding adenovirus) or, as a negative control, vLA-1 (CCR5-encoding adenovirus) at a moi of 3 pfu/cell. Infected cells were incubated for two days to allow expression of recombinant proteins then infected with either an X4 or a R5 HIV-1. Under these experimental conditions, a pronounced reduction in HIV-1 productive infection was observed (>50% with IIIB Vs 40% with Ba-L; Figure 18).
  • the ⁇ 32 was delivered to PBMCs using Ad5/ ⁇ 32 at 3 pfu/cell and a 50% reduction in infectivity was observed.
  • the efficiency adenovirus vector depends highly on the kind of cells used and a careful standardization of gene delivery into the PBMCs is necessary to perform in order to determine whether full resistance to infection can be obtained with higher doses of ⁇ 32.
  • Figure 18 shows the Phytohemagglutinin-A+IL2-activated Ficoll-purified human PBMCs which were infected with either vLA-1 (Ad5/CCR5) or vLA-2 (Ad5/ ⁇ 32) at 3pfu/cell for each virus for two days then infected with either Ba-L (R5) or IIIB (X4). Infections were performed in a 96-well plate. The virus was absorbed for three hours and cells were washed three times with PBS and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100mU of recombinant IL-2 and 10mg/ml PHA.
  • Culture fluid 50 ⁇ l was harvested after cell resuspension every three days and replaced with fresh medium.
  • the amount of p24 antigen in the cell- containing supernatants was measured using an Elisa kit purchased from DuPont.
  • the AZT control infection resulted in p24 values below 0.5 ng/ml.
  • Figure 19 shows the model for the ⁇ 32 effect.
  • ⁇ 32 protein interacts specifically with the major HIV coreceptors interfering with their proper transport to the cell surface.
  • the impairment of coreceptor molecules reduces their chance to associate with CD4 leading to unfavorable stoichiometry for virus entry.
  • the ⁇ 32 effect is concentration-dependent. Higher ⁇ 32 levels can correlate with less coreceptor and reduced virus entry. This concentration effect explains why +/- carriers show partial protective phenotype.
  • Infected -/- cells either expresses low levels of ⁇ 32 or expresses a defective ⁇
  • the data provides evidence for the ⁇ 32 effect on X4 fusion/infection.
  • This experiment examined whether ⁇ 32 protein colocalizes with the major coreceptors and provide evidence for their physical association using protein heterodimerization, co-immunoprecipitation and the yeast 2-hybrid system. These experiments set the stage for experiments focused at analyzing the structural determinants involved in ⁇ 32-coreceptor association. Many integral membrane proteins form noncovalently associated oligomeric complexes that are prerequisites for transport and normal function.
  • the present study is the first to utilize the ⁇ 32 open reading frame containing the frame shift 31 amino acid residues that are not expressed in CCR5.
  • Confocal microscopy was used to detect ⁇ 32-coreceptor complexes using fluorescently labeled molecules.
  • Cells expressing endogenous CCR5 and CXCR4 i.e. MAGI-CCR5 cell line
  • Ad5/ ⁇ 32 was infected with Ad5/ ⁇ 32, examined for the ⁇ 32 effect (downmodulation effect, as in Figure 4) and used to examine whether ⁇ 32 colocalizes with CCR5 or CXCR4.
  • Cells were either fixed or permeablized, and incubated with anti- ⁇ 32 and FITC-labeled secondary antibody.
  • Cell samples were then reacted with either anti-CCR5 or anti-CXCR4 monoclonal antibodies and a phycoerythrin-labeled secondary antibodies. Confocal laser scanning microscopy was performed to detect colocalization of the proteins.
  • Recombinant Ad5 encoding ⁇ 32 or either of the major coreceptors was used to co-infect NIH-3T3 cells.
  • total cell lysates were prepared and immunoprecipitated using the anti-CXCR4 antisera (Feng, et al.) or anti-CCR5 antibodies. Immnuoprecipitation with an antibody followed by western blotting with a different antibody provides a way to determine whether hetero- oligomers form.
  • the immunoprecipitates were fractionated in 10%SDS-PAGE and transferred onto nitrocellulose membranes.
  • the blots were initially probed with anti- ⁇ 32 antibodies to verify the presence of ⁇ 32 protein, then stripped and reprobed with an antibody specific to either CXCR4 or CCR5. Since the ⁇ 32 protein has a lower molecular weight (28-30KD) compared to CXCR4 or CCR5 (45-50KD), it was possible to distinguish the ⁇ 32-CXCR4 or ⁇ 32-CCR5 heterodimers from the CXCR4 or CCR5 homodimers on the immunoblot. Monoclonal antibodies specific to ⁇ 32 protein helped overcome some of the technical difficulties associated with the detection of ⁇ 32-CXCR4 complexes. Co- immunoprecipitation was used as an alternative approaches to confirm the existence of ⁇ 32-coreceptor complexes.
  • Cells co-expressing ⁇ 32 and CCR5 or CXCR4 (recombinant or endogenous) was used for immunopricipitation with anti- ⁇ 32 antibodies.
  • the immune complexes were fractionated and then immunoblotted.
  • Co- precipitated CCR5 or CXCR4 was detected using specific antibodies to either protein.
  • Yeast two-hybrid systems provide a sensitive method for detecting transient protein interactions that are biochemically detectable.
  • the system sensitivity allows quantitative analysis of mutant constructs to be used in the mapping studies.
  • the system has been previously used to analyze CCR5 truncated molecules with wild- type CCR5 (Benkirane, et al.).
  • a MATCHMAKER two-hybrid system purchased from CLONTECH was used to analyze ⁇ 32-co receptor interaction.
  • the cDNA encoding ⁇ 32 ORF was cloned into pGBKT7 (CLONTECH) while the coreceptor cDNA was cloned into pGADT7 (CLONTECH).
  • Yeast AH109 (or Y187) was cotransformed with pGBKT7- ⁇ 32 and pGADT7-coreceptor.
  • the cotransformation mixture was plated on SD/-Leu/-Trp to select for colonies containing both hybrid plasmids.
  • a colony-lift ⁇ -galactosidase assay was used as the system PHS 398.
  • X-gal staining of transformed colonies detects protein interaction. Quantitative analysis of the interaction was performed using 5-bromo-4-chloro-3-indolyl- ⁇ -D- galactopyranoside as a substrate in liquid cultures. The amount of ⁇ -galactosidase made as a result of protein/protein interaction was quantitated using a colorimetric method. Parallel cotransformation controls using pGBKT7- ⁇ 32 and either pGADT7- CXCR2, pGADT7-CD4, or pGADT7-CD44 was performed to control for the background of the system. The preliminary data indicated that ⁇ 32 has no downmodulation effect on CD4, CXCR2 or CD44 ( Figurel O) and therefore, was used as negative controls for coreceptor- ⁇ 32 interactions.
  • ⁇ 32 protein interacts with and specifically inactivates functional expression of other HIV coreceptors.
  • CCR5 and CXCR4 are the major HIV coreceptors
  • CCR3 plays a role in HIV pathogenesis of the brain (He, et al.).
  • Possible contribution of other coreceptors to HIV pathogenesis of other tissues has been recently reported (Lee, et al., Sharron, et al., Faure, et al.).
  • the result of this analysis explains why other coreceptors in -/- individuals are not utilized in the absence of CCR5.
  • the effect of ⁇ 32 on nonhuman CXCR4's were examined to analyze the specificity of ⁇ 32 activity.
  • CD4+ NIH-3T3 cells were coinfected with recombinant adenoviruses encoding ⁇ 32 and the appropriate coreceptor (or a chemokine receptor) and Ad5pol3 (T7 RNA Polymerase). Effector Hela cells was coinfected with vCB-21 R (T7lacZ) and one of the HIV-1 Envs-expressing vaccinia vectors.
  • the recombinant adenovirus expressing ⁇ 32 protein was used as an antigen to immunize the mice. This provides a method to deliver ⁇ 32 in its native conformation. These experiments take place in the animal colony of Indiana University medical center.
  • the generated MAbs was used as reagents to detect ⁇ 32, and to map its functional domain(s) using a panel of point mutants. Since Mabs were generated using the vLA-2 that delivers native ⁇ 32, generated antibodies had different specificities that helped map critical epitopes in ⁇ 32. This was accomplished by examining the ability of ⁇ 32 variants (chimeras and point mutants) to dbwnmodulate coreceptors and be recognized by a particular Mab.
  • ⁇ 32 protein impairs the formation of CD4-Coreceptor complexes by reducing coreceptor availability.
  • Coreceptors compete for association with CD4 (Lee, et al.) ⁇ 32 protein reduces this interaction by associating with the coreceptor.
  • CD4-coreceptor complexes exist without the presence gp120 and by using confocal microscopy the colocalization of such complexes has been demonstrated
  • CD4-CCR5 complexes are stronger than CD4-CXCR4 complexes. It is not known however, whether CD4- coreceptor association occurs cotranslationaly or at the cell surface.
  • Hela cells were used in this analysis because it expresses endogenous CXCR4.
  • a CD4-encoding vaccinia virus using different doses of the virus delivers increasing levels of CD4. These expression levels were established by infecting Hela cells with 1 pfu/cell (low), 5 pfu/cell (medium) or 20pfu/cell (High). Cells were verified for CD4 expression by quantitative FACS analysis.
  • the ⁇ 32 protein was introduced into these cells by Ad5/ ⁇ 32 at an inhibitory dose (10-20pfu/cell) and challenged with X4 or R5 Env-expressing cells. The effect of high CD4 levels delivered by the vaccinia vector was assayed by quantitating X4 and R5 fusion compared to control virus-infected cells.
  • CD4 associates with coreceptor cotranslationally, then it is possible to colocalize the CD4-co receptor ⁇ 32 by confocal microscopy.
  • a similar approach was taken to study the effect of CCR5 and CXCR4 concentrations on ⁇ 32 effect in cells expressing limited amounts of CD4. This analysis determines whether the ratio of pre-existing CD4-CCR5 to CD4-CXCR4 complexes influences ⁇ 32 effect.
  • Cell lines such as MAGI-CCR5 were suitable for this kind of analysis since it contains all molecules involved in the process.
  • the levels of CD4 expressed by vaccinia virus vectors are very high and may not be relevant to the in vivo situation. Additionally, high multiplicity infection can change membrane structures leading to high background levels.
  • cell lines expressing different levels of CD4 were used and assayed for its effect on intracellular ⁇ 32.
  • CCR2 shares more than 75% homology with CCR5 Alkhatib, et al., it was not downmodulated by ⁇ 32 suggesting a possible specific interaction that involves at least, part of the non-homologous region.
  • ⁇ 32 expression has no significant effect on surface levels of CD4 confirming that the observed reduction in X4 fusion/infection is not due to an effect on CD4.
  • CCR5/CCR2 hybrid constructs and CCR5 point mutants were also utilized to analyze the region of CCR5 that is likely to interact with ⁇ 32.
  • CXCR4 point mutants were utilized to determine a potential region of CXCR4 that interacts with ⁇ 32.
  • the unique carboxy terminal 31 amino acids in the ⁇ 32 protein create new functional domain(s) that can be specific for interaction with coreceptors.
  • a direct way to determine whether the carboxy terminal region of ⁇ 32 is critical for its activity is to construct chimeric molecules where this unique ⁇ 32- specific COOH tail or portion of it is grafted onto CCR5 that lacks its carboxy tail.
  • CCR5 lacking the carboxy terminal cytoplasmic tail is expressed at the cell surface and is functional as a coreceptor (Alkhatib, et al.).
  • Co-expression of tail-less CCR5 and wild type CCR5 or CXCR4 had no effect on R5 or X4 fusion, which is in agreement with published data using a similar construct (Benkirane, et al.).
  • mutants can be designed to include the shared homologous region with CCR5.
  • CXCR4 and CCR5 share only 30% homology (including conserved amino acid changes), it is important to examine whether a common region of the principal coreceptors CXCR4 and CCR5 is responsible for interacting with ⁇ 32. Knowledge of this common region provides insight into the molecular basis of coreceptor activity and the design of new drugs that can have a broader antiviral effect.
  • a direct method to map important regions of coreceptor involved in interaction with ⁇ 32 can be to construct chimeric molecules between CXCR4 or CCR5 with homologs that do not function as coreceptors or show a different coreceptor function in terms of Env specificity (i.e. CCR2 works only with 89.6 but not with R5 Envs).
  • the hybrid molecules are transfected into 293 cells followed by infection with Ad5/ ⁇ 32 and FACS analysis to determine the hybrid molecules that lost the ability to associate with ⁇ 32.
  • the loss of function is characterized by the ability of the hybrid molecule to escape the effect of ⁇ 32 indicative by its efficient cell surface expression.
  • Obtaining functional mutants is also useful to verify and confirm the interactive region.
  • CXCR4 point mutants provided by Chris Broder (Chabot, et al.)
  • CCR5 point mutants Alkhatib, et al.
  • Most CXCR4 point mutants are expressed at the cell surface and therefore, are assayed for the ⁇ 32 effect by FACS analysis.
  • Antibodies to ⁇ 32 are now available and are being used to specifically detect ⁇ 32 protein in Western blots and immunoprecipitation. One first determines whether ⁇ 32 physically associates with the major coreceptors. The biological analyses of chimeric mutants as well as the heterodimer formation experiments can initially use the polyclonal antibodies for detecting the mutant protein. Once the Mabs are made they are used with ⁇ 32 chimeras and point mutants to map the critical determinants of this protein.
  • CCR5/ ⁇ 32 heterozygosity confers a different degree of protection against HIV-1 in PBLs and MDMs, depending on the ratio of wild-type and mutant ⁇ 32 mRNA in the two cell types.
  • the investigators analyzed expression levels of wild type and mutant ⁇ 32 mRNA species by competitive RT-PCR and demonstrated that depending on the cell type, ⁇ 32 heterozygosity confers a different degree of protection against HIV-1 infection.
  • Antibodies raised against the COOH-terminus of ⁇ 32 to analyze its expression in PBMCs of -/- and +/- individuals were used.
  • a number of -/-, +/-, and +/+ PBMCs are used.
  • Cell lysates of unstimulated PBMCs, or PHA-stimulated PBMCs from homozygotes and heterozygotes are prepared and analyzed for expression of endogenous ⁇ 32 protein by immunoblotting and immunoprecipitations as presented in the preliminary data section. Quantitative protein analysis are performed to determine whether there are differences in ⁇ 32 protein levels in -/- versus +/- individuals that can explain the partial, incomplete protective phenotype in heterozygotes. Similar analysis are performed on the infected -/- PBMC sample to determine whether ⁇ 32 protein is expressed in these cells.
  • PBMCs from one infected ⁇ 32 homozygote is not representative of all 8 homozygotes identified so far (Michael, et al.). Unfortunately, some of these individuals are dead. and it is possible to examine their samples.
  • PBMCs from -/- individuals provide an in vivo system where the effect of ⁇ 32 on endogenous coreceptors can be assayed in their native environment.
  • ⁇ 32 protein is expressed and can be detected in -/-PBMCs ( Figure 15). It is important to examine ⁇ 32-CXCR4 interaction in -/- PBMCs to provide an in vivo evidence for this molecular association. As mentioned earlier, the association of ⁇ 32 and CXCR4 is relevant to pathogenesis.
  • Pulse chase experiments are performed to compare coreceptor protein synthesis in -/- and +/+ PBMCs to directly examine biosynthesis and stability of coreceptor proteins made in these two cell populations. Unstimulated as well as PHA+IL2 stimulated -/- and +/+ PBMCs are used for labeling with 35 S-methionine.
  • Activated cells express higher levels of endogenous proteins and therefore can be useful in tracing coreceptor proteins in pulse-chase experiments.
  • the protein analysis shown in Figure 15 indicated the detection of ⁇ 32 protein in unstimulated cells.
  • cell lysates are prepared and used in co-immunoprecipitation assays.
  • the effect on stability of CXCR4 in -/- versus +/+ PBMCs is examined in pulse chase experiments followed by immunoprecipitation of CXCR4 with monospecific antibodies (Feng, et al.).
  • the experiment is focused on developing a cellular model system to examine other factors that can contribute to resistance to HIV-1. Identification of these factors opens new avenues in this field and has important implications for understanding the molecular basis of resistance to HIV-1.
  • the human myeloid HL-60 cell line can be differentiated into macrophages upon treatment with RA.
  • RA treatment Upon RA treatment, these cells differentiate to acquire a macrophage phenotype and become fusogenic with R5 tropic HIV-1 (Alkhatib, et al.). Since the ⁇ 32 effect is observed in HL60 differentiated cells, then it suggests expression of another cellular protein that can be critical for the ⁇ 32 effect.
  • ⁇ 32 can downmodulate CXCR4 in RA-treated HL60 cells using untreated cells as a negative control.
  • Subtraction cDNA library is made using undifferentiated and RA-differentiated cells as a source of RNA.
  • the induced molecule(s) is required for ⁇ 32 then it can interact with it.
  • a direct method to examine and isolate the interacting protein is using the yeast two-hybrid screening system. Once the cDNA is/are isolated, it is transfected into untreated HL60 cells to examine if it can transfer the ⁇ 32 effect.
  • the cDNA clone is sequenced and its identity and homology with other proteins are analyzed.
  • the induced molecules in RA-treated HL60 cells cannot interact with ⁇ 32 but can interact with CXCR4.
  • Another two-hybrid screening is performed using CXCR4 as the bait protein. If these screening methods failed to identify the induced molecule, a functional cDNA screening method is used to isolate the cDNA clone. This method is based on the transfer of ⁇ 32 function to untreated HL60 cells. Briefly, the cDNA library under T7 promoter is transfected into HL60 cells, followed by infection with Ad5pol3 (T7 RNA pol) and Ad5/ ⁇ 32. Reduction in X4 fusion indicates transfer of function. The cDNA library is then subfractionated and fractions are used to repeat the screening until a single clone is identified.
  • Cell lines transformed with ⁇ 32 provide a cellular model for the analysis of ⁇ 32 activity and its contribution to resistance to HIV-1. Creation of this cell line is critical to analyze ⁇ 32-CXCR4 complex formation in cells synthesizing endogenous levels of ⁇ 32 and CXCR4 proteins. This is a ⁇ 32-CXCR4 interaction without the presence of endogenous CCR5. This allows studying the effect of introduction of exogenous CCR5 in order to determine whether the major coreceptors compete for complexing with ⁇ 32. The analysis of ⁇ 32 has relied on Ad5 vectors to express recombinant ⁇ 32 in cell lines as well as primary cells. In all experiments the ⁇ 32 gene was introduced into cells expressing endogenous CXCR4 and/CCR5.
  • FIG. 17 and 18 The preliminary data shown in Figures 17 and 18 indicated that cell lines as well as PBMCs expressing recombinant ⁇ 32 showed reduced susceptibility to HIV-1 infection. It is of basic interest to examine ⁇ 32 effect in a CD4+ T-cell line that expresses endogenous levels of ⁇ 32 protein.
  • Jurkat cells are CD4+CXCR4+ and endogenous expression of ⁇ 32 directly address its effect on endogenous CXCR4 expression and susceptibility to infection. This allows detailed analysis of the intracellular distribution and localization of ⁇ 32 protein and its possible interaction with CXCR4.
  • the choice of Jurkat cell line is based on the fact that it is CD4+CXCR4+ and it shows ⁇ 32 effect when infected with Ad5/ ⁇ 32.
  • Standard procedures are used as previously described to create a Jurkat- CCR5 cell line (Alkhatib, et al.). Positive clones are identified by PCR on cellular genomic DNA. Clones are selected on the basis of the ⁇ 32 made using RT-PCR as a method of quantification. Clones with low, medium, and high levels of ⁇ 32 mRNA expression are selected and expanded. Antibodies to ⁇ 32 can finally be used to identify the clones expressing low, medium, or high levels of the ⁇ 32 protein. Once clones of ⁇ 32 cell lines are purified they can be tested in infectivity assays to determine the percent reduction in HIV-1 fusion/infection assays. The selected clones expressing different levels of ⁇ 32 are extremely useful in determining whether a dose-effect correlation can exist in vivo.
  • Example 2 The Yeast Two-hybrid system was used to provide evidence for CXCR4 interaction with the Delta-32 protein.
  • GAL4-based Matchmaker two-hybrid system 3
  • the system provides a transcriptional assay for detecting protein interactions in vivo in yeast.
  • Delta-32 gene was expressed as a fusion to the GAL4 DNA-binding domain (DNA-BD), while CCR5 or CXCR4 was expressed as a fusion to the GAL4 activation domain (AD).
  • DNA-BD fusion vector, pGBKT7 and AD fusion vector pGADT7 were used for high level expression.
  • Delta-32 (cloned in pGBKT7) and CCR5/CXCR4 (cloned in pGADT7) inserts were expressed as GAL4 fusions with c- Myc and hemagglutinin (HA) epitope tags, respectively.
  • the transcription and translation of epitope-tagged fusion proteins in vitro was driven by T-7 promoter, which is at the downstream of the GAL4 coding sequence.
  • Cotransformation was carried out using both the bait -Delta-32 and AD fusion vector-CCR5/CXCR4 in yeast strain AH 109, which is gal4 " and gal80 " prevents interface of native regulatory proteins with the regulatory elements in the two-hybrid system.
  • pGBT7-53 and pGADT7-T encode fusions between the GAL4 DNA-BD and murine p53 and SV40 large T-antigen, respectively.
  • p53 and large T-antigen interact in a yeast two-hybrid assay and was used as positive control.
  • PGBKT-7-Lam encodes a fusion of the DNA-BD with human lamin C and provides a control for a fortuitous interaction between an unrelated protein and the pGADT7-T and was used as a negative control.
  • Exp.#1 is a negative control.
  • Exp.#2 is a positive control for interaction of two well-known proteins.
  • Exp. #3, 4, and 5 are all controls to measure the background of the system.
  • Exp.#6 and 7 represent confirmed results of ⁇ -32 protein interaction with not only CCR5 but also CXCR4.
  • Interaction of ⁇ -32 with CXCR4 is a novel finding that has never been described in the field of AIDS. This finding is significant because CXCR4 is linked to disease progression and represent an important coreceptor that the majority of HIV-1 isolates utilize during the symptomatic stage of AIDS.
  • Example 3 Example 3:
  • HIV-1 Human immunodeficiency virus type 1
  • CD4 and a coreceptor, principally the CCR5 and/or CXCR4 chemokine receptors, for entry into host cells.
  • CCR5 The central role of CCR5 in HIV-1 transmission and pathogenesis has been highlighted by the epidemiological and genetic identification of powerful disease-modifying effects of the naturally occurring CCR5 ⁇ -32 (D32) allele, a 32- base pair deletion encoding a truncated and non-cell surface expressed version of CCR5. Relative to the general population, D32/D32 homozygotes are rarely found among HIV+ individuals but are significantly more common among repeatedly exposed/uninfected (EU) individuals.
  • EU repeatedly exposed/uninfected
  • D32 delta-32
  • ORF open reading frame
  • HIV-1 Human immunodeficiency virus type 1
  • CD4 and a coreceptor, principally the CCR5 and/or CXCR4 chemokine receptors, for entry into host cells.
  • the central role of CCR5 in HIV-1 transmission and pathogenesis has been high-lighted by the epidemiological and genetic identification of powerful disease modifying effects of the naturally occurring CCR5 ⁇ 32 allele, a 32 base pair deletion encoding a truncated and non-cell surface expressed version of the coreceptor.
  • ⁇ 32 Delta-32
  • ORF open reading frame
  • ⁇ 32 protein expression can specifically reduce the expression of endogenous CCR5 or CXCR4 resulting in the inhibition of virus entry and infection by R5 and X4 HIV-1 isolates. This effect was not observed in cells expressing recombinant CCR5 or other non-relevant proteins such as measles virus hemaglutinin (MVHA) or structural adenovirus proteins.
  • MVHA measles virus hemaglutinin
  • ⁇ 32 protein down-regulates the major coreceptors resulting in an unfavorable stoichiometry of the molecules involved in viral entry.
  • ⁇ 32-specific antibodies we have shown that ⁇ 32 is expressed in ⁇ 32/ ⁇ 32 PBMCs.
  • HIV-1 human immunodeficiency virus type 1
  • CD4 human immunodeficiency virus type 1
  • CXCR4 the major coreceptors used by most R5 and X4 HIV-1 isolates.
  • the importance of chemokine receptors in HIV-1 transmission is highlighted by the finding that individuals homozygous for a 32-base pair deletion in CCR5 ( ⁇ 32/ ⁇ 32) are resistant to HIV-1 infection.
  • the defective coreceptor gene encodes a prematurely terminated protein (Fig.lA) that is not detected at the cell surface and therefore is not functional as a fusion coreceptor.
  • ⁇ 32 homozygous as -/-
  • ⁇ 32 heterozygous as +/-
  • Genotypic analysis of this mutation and its distribution revealed that ⁇ 32 has a high allele frequency among Caucasians but was absent in African or Asian populations.
  • the mutant allele is not associated with any obvious phenotype in uninfected homozygous individuals.
  • Heterozygotes (+/-) are not protected against infection, but once they become infected, have a slower progression to AIDS, indicating that partial resistance can occur in the presence of a single copy of the mutant CCR5 gene.
  • the frameshift ⁇ 32 mutation introduces 31 new amino acid residues at the carboxy terminus of ⁇ 32 that are not present in CCR5 (Fig. 1A&C).
  • the CCR5 ⁇ 32 protein was expressed and analyzed in 293 cells infected with a recombinant adenovirus Ad5/ ⁇ 32 ( Figure 1 B) (which also express green fluorescent protein) using a 31 -amino acids custom peptide antibody generated against the carboxy terminus of ⁇ 32-ORF ( Figure 1C).
  • the ⁇ 32 protein was found to be abundantly expressed in 293 cells infected with Ad5/ ⁇ 32 and showed a protein band at around 28-30 kDa ( Figure 2A &B)).
  • the ⁇ 32 protein was also detected using anti-CCR5 antiserum directed against the common N-terminus of CCR5 and CCR5 ⁇ 32.
  • immunostaining of ⁇ 32 protein indicated that it is expressed intracellularly towards the inner side of the plasma membrane ( Figure 2C).
  • PBMCs freshly isolated from healthy donors.
  • PBMCs were stimulated with PHA+IL-2 for 3 days then used for FACS staining and cell fusion.
  • the PBMCs were infected with Ad5/ ⁇ 32 and examined for ⁇ 32 effect on cell surface levels of CCR5, CXCR4, CD4, CD25, CD44, and CXCR2 by FACS analysis.
  • Ad ⁇ MVHA, or wild type Ad5 vector was included infected with Ad5/CCR5.
  • the degree of fusion inhibition was proportional to the amount of expressed ⁇ 32 protein made in the infected PBMCs ( Figure 3, protein blot).
  • the gradual increase in the inhibitory effect was proportional to the increasing intensity of ⁇ 32 protein band.
  • we found that the levels of ⁇ 32 protein obtained by our AD ⁇ / ⁇ 32 could not be obtained by simple DNA transfection of 293 cells (data not shown) which may explain the inability of Venketesan, et al. (Bleul, et al.) to observe the ⁇ 32 effect.
  • RNA expression of ⁇ 32 was verified by RT-PCR analysis ( Figure 6A).
  • the immunoblot analysis revealed a protein band (Figure 6B&C) that corresponds to the same molecular weight band seen with recombinant protein analysis ( Figure 2). These protein bands were not obtained with the preimmune serum. The identity of the band that appears above the 34 Kd marker band is not known at present, however, since it is expressed in normal CCR ⁇ individuals, it could represent a cross- reactive cellular protein.
  • Our analysis also revealed that the ⁇ 32 protein band detected in the protected (-/-) individuals was absent in an infected (-/-) individual ( Figure 6C) suggesting a critical role for the ⁇ 32 ORF in resistance to HIV.
  • Recombinant ⁇ 32 conferred a broad protective effect against X4 and R ⁇ HIV- 1 infection.
  • ⁇ 32 protein has a lower molecular weight (28-30kDa) compared to CXCR4/CCR ⁇ (45- 50KD), it will be possible to distinguish the ⁇ 32-CXCR4 or ⁇ 32-CCR ⁇ heterodimers from the CXCR4 or CCR ⁇ homodimers on the immunoblot.
  • Anti-c-myc or anti-HA antibodies will also be used for immunoprecipitating ⁇ 32 with CXCR4 or CCR5 in yeast two hybrid system as ⁇ 32 and the coreceptors are expressed as fusion proteins of c-myc and HA.
  • polyclonal antibodies to specifically detect recombinant and native ⁇ 32 protein.
  • Monoclonal antibodies to ⁇ 32 protein may help overcome some of the technical difficulties associated with the detection of ⁇ 32- CXCR4 complexes.
  • Co-immunoprecipitation will be used as an alternative approaches to confirm the existence of ⁇ 32-coreceptor complexes.
  • Cells co- expressing ⁇ 32 and CCR ⁇ or CXCR4 (recombinant or endogenous) will be used for immunoprecipitation with anti- ⁇ 32 antibodies.
  • the immune complexes will be fractionated and then immunoblotted.
  • Co-precipitated CCR ⁇ or CXCR4 will be detected using specific antibodies to either protein.
  • the recombinant adenovirus Ad ⁇ / ⁇ 32 is used as an antigen to immunize the mice. These experiments take place in the animal colony of Indiana University medical center. This will deliver the ⁇ 32 protein in its native conformation providing an opportunity to generate Mabs to native ⁇ 32 epitopes. Examining the ability of ⁇ 32 variants (chimeras and point mutants) to downmodulate coreceptors and be recognized by a particular Mab will help generate a functional map of ⁇ 32. By generating a series of Mabs and continuing these downmodulation experiments we will be able to generate a functional map of ⁇ 32.
  • Subcellular localization For intracellular co-localization studies, paraformaldehyde-fixed cells will be washed once with HBSS/BSA and then incubated in HBSS/BSA containing 0.0 ⁇ % saponin for 30 min at room temperature.
  • ER endoplasmic reticulum
  • PDI protein disulfide isomerase
  • a direct way to determine whether the carboxy-terminal region of ⁇ 32 is critical for its activity will be to construct chimeric molecules where this unique ⁇ 32- specific COOH tail or portion of it is grafted onto CCR ⁇ that lacks its carboxy tail (gain of ⁇ 32 function).
  • CCR ⁇ lacking the carboxy terminal cytoplasmic tail is expressed at the cell surface and is functional as a coreceptor. If the biological activity of ⁇ 32 is contained within the last 31 amino acids at its carboxy terminus, then adding this region to a tail-less CCR ⁇ is expected to result in the loss of its ability to reach the cell surface. This analysis will be extended to include other truncation of this ⁇ 32 fragment in order to map its interactive domain.
  • the ⁇ 32 may be used as a therapeutic approach to induce resistance to HIV-1 infection.
  • Gene delivery of the ⁇ 32 to stem cells may provide the opportunity of producing progeny cells that resist HIV-1 infection.
  • the ⁇ 32 protein is a naturally expressed molecule and individuals.- carrying this mutation do not show any obvious hematopoietic defects or any other immunological disorders.
  • CCR3 and CCR ⁇ are co-receptors for HIV-1 infection of microglia," Nature, 38 ⁇ (6617) 64 ⁇ -9 (1997).
  • CCR5 coreceptor usage of non-syncytium-inducing primary HIV-1 is independent of phylogenetically distinct global HIV-1 isolates: delineation of consensus motif in the V3 domain that predicts CCR-5 usage," Virology, 240(1), 83-92 (1998).
  • Alkhatib, G. et al. "High-level eucaryotic in vivo expression of biologically active measles virus hemagglutinin by using an adenovirus type 5 helper-free vector system," J. Virol., 62, 2718-2727 (1988).
  • Alkhatib, G., et al. "Expression of bicistronic measles virus P/C mRNA by using hybrid adenoviruses: levels of C protein synthesized in vivo are unaffected by the presence or absence of the upstream P initiator codon," Journal of Virology, 62(1 1 ), 4059-69 (1988).
  • HIV-1 Co-receptor activity share common structural determinants: Critical residues in the third extracellular loop support HIV-1 fusion," Journal of Biological Chemistry, 272(32), 19771 -6 (1997).

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Abstract

There is provided a method of protecting individuals from contracting HIV by administering either a vector or a vaccine containing a sequence encoding the Δ32 mutation. Also provided is a method of decreasing the amount of HIV co-receptors present on the cell surface by administering a compound including a sequence encoding the Δ32 mutation in a pharmaceutically acceptable carrier. Also provided is a compound for decreasing the amount of HIV co-receptors present in the cell surface, the compound having a sequence encoding the Δ32 mutation in a pharmaceutically acceptable carrier. Also provided is a vector containing a sequence encoding the Δ32 mutation. An assay for testing the efficacy of HIV treatment is also provided, the assay includes a detector for detecting the presence of the Δ32 mutation in cells.

Description

TREATMENT AND PREVENTION OF AIDS PROGRESSION AND METHODS OF USING SAME
BACKGROUND OF THE INVENTION
1. FIELD OF THE INVENTION
The present invention relates to methods and compounds for treating HIV. More specifically, the present invention relates to methods of preventing the transmission and progression of AIDS.
2. DESCRIPTION OF RELATED ART
HIV-1 is the causative agent of acquired immunodeficiency syndrome (AIDS). The virus specifically targets helper T lymphocytes and cells of the monocyte- macrophage lineage through high affinity interaction with the CD4 surface antigen (reviewed in Berger, et al., Broder, et al.). Various studies indicated that CD4 alone was not sufficient for HIV-1 entry and that another cofactor was essential for fusion/infection). A major discovery in 1996 concluded that the second receptor (coreceptor) used by HIV-1 belonged to a large family of seven transmembrane G- protein coupled receptors (Feng, et al.). The CXC chemokine receptor CXCR4 is the coreceptor used by X4 HIV-1 isolates (Feng, et al.) while the CC chemokine receptor CCR5 serves as the coreceptor for R5 HIV-1 strains (Doranz, et al., Choe, et al., Alkhatib, et al., Deng, et al., Dragic, et al.).
The membrane orientation model of the 7TM domains chemokine receptor proteins (CCR5 is an example) is known to those in the art. The amino acid sequence of the translated protein predicts seven transmembrane domains, an extracellular amino terminus and three extracellular loops, an intracellular carboxy terminus, three intracellular loops, and a S-S-proposed disulfide bond. The conserved serine/Threonine rich cytoplasmic tail is the proposed domain involved in G-protein-coupled signaling. Chemokine receptors share an overall 30% amino acid homology. Members of the same family share a higher degree of homology. In addition to CXCR4 and CCR5, the major coreceptors for TCL-tropic and M-tropic HIV-1 , and other chemokine receptors such as CCR2b (Doranz, et al.), CCR3 (Doranz, et al., Alkhatib, et al., Choe, et al.), CCR8 (Rucker, et al.), STRL33 (BONZO) (Liao, et al., Deng, et al.) BOB (GPR15) (Deng, et al.) were also found to serve as fusion cofactors for HIV-1 entry. Despite the large number of related molecules showing an ability to employ alternate coreceptors in entry and infection, the principal coreceptors remain the initially discovered CXCR4 and CCR5 molecules.
It is well documented that HIV-1 binds to CD4 via an interaction between the first domain of CD4 and a discontinuous region of the external subunit of HIV-1 envelope glycoprotein, gp120. This region is referred to as the CD4 binding site (reviewed in Moore, et al.). An accepted model for viral entry states that gp120 subunit binds first to CD4 resulting in a conformational change followed by interaction with the coreceptor which leads into another conformational change that exposes the fusion peptide of gp41 initiating the process of membrane fusion (reviewed in Moore, et al.).
A detailed analysis of CD4-CCR5 interaction demonstrated that the concentrations of CD4 and CCR5 required for efficient R5 infection are interdependent and that the requirements for each are increased when the other component is present in a limiting amount (Platt, et al.). Recent work demonstrated that R5 infection requires the concerted actions of multiple CCR5 molecules and provided a mathematical model where 4-6 CCR5 monomers can be involved during R5 entry (Kuhmann, et al.). Similar studies have demonstrated that CXCR4 can have the same requirement to form a complex that is essential for X4 infection (Dimitrov, et al.). Although these studies suggest physical interaction between CD4- coreceptor, the optimal stoichiometry of this association remains unknown.
Discovery of the naturally occurring Δ32 mutation and its association with resistance to HIV-1 was previously known. Genotypes are represented by (+) for a wild-type allele and (-) for a Δ32 allele. Δ32 homozygotes are referred to as -/-, Δ32 heterozygotes as +/-, and to those with wild type CCR5 as +/+.
The amino acid structure of the Δ32 mutant protein has been predicted based on the transmembrane structure of the wild type CCR5 sequence. The Δ32 deletion is located in a region corresponding to the second extracellular loop and results in a frameshift that produces a smaller protein which lacks the last three transmembrane domains and the carboxy terminal tail involved in G-protein signaling. The frameshift caused by Δ32 deletion introduces 31 new amino acid residues that are not encoded by CCR5. The importance of chemokine receptors in HIV-1 transmission is highlighted by the finding that individuals homozygous for a 32-base pair deletion in CCR5 (Δ32/ Δ32) are resistant to HIV-1 infection. The deletion resulted in a frameshift mutation that introduced 31 new amino acid residues at the carboxy terminus of Δ32 that are not present in CCR5. The defective coreceptor gene encodes a prematurely terminated protein that is not detected at the cell surface and therefore is not functional as a fusion coreceptor (Samson, et al., Dean, et al., Huang, et al., Zimmerman, et al., Liu, et al.). Genotypic analysis of this mutation and its distribution revealed that Δ32 has a high allele frequency among Caucasians but was absent in African or Asian populations (Samson, et al., Dean, et al., Zimmerman, et al., Liu, et al.). The mutant allele is not associated with any obvious phenotype in uninfected homozygous individuals. Heterozygotes (CCR5/Δ32) are not protected against infection, but once they become infected, have a slower progression to AIDS (Samson, et al., Dean, et al., Zimmerman, et al., Liu, et al.), indicating that partial resistance can occur in the presence of a single copy of the mutant CCR5 gene.
Although homozygosity for Δ32 mutation is associated with disease resistance, HIV-1 infection has been reported in one person with hemophilia (O'Brien, et al.) and several -/- homosexuals Balotta, et al., Biti, et al., Theodorou, et al.). The reported number of infected Δ32 homozygotes is currently 8 (reviewed in (Michael, et al). None of the reports, which documented infection of Δ32 homozygotes analyzed expression of native Δ32 protein in primary cells of infected individuals. Endogenous levels of Δ32 mutant protein have never been verified in these patients or in any homozygote. The expression of the Δ32 protein was analyzed in two brothers that are homozygous for the Δ32 allele; one infected and the other uninfected. The results confirmed that the protected brother expressed Δ 32 protein whereas the infected brother lacked such expression. The absence of Δ 32 protein expression in this infected homozygote implicates a critical role for the protein in resistance to HIV-1. A growing body of evidence suggests that Δ32 heterozygosity is associated with reduced risk to some complications of the disease. For example, studies analyzing the effect of +/- genotypes indicated reduced prevalence of Δ32 mutation in those who do develop AIDS dementia complex (ADC) (van Rij, et al.). Others reported an association of Δ32 mutation with protection against HIV-associated lymphoma (Dean, et al.). Recent reports described that carriers of the Δ32 mutation respond better to highly active antiretroviral therapy (HAART) treatment compared to wild type controls (O'Brien, et al., Workman, et al.). Moreover, CD8+ T cells purified from +/- individuals showed increased CTL activity compared to normal controls (Connick, et al.).
The first attempt to shed a light on the mechanism of resistance to HIV-1 observed in Δ32/Δ32 carriers postulated that protection is due to increased levels of CC chemokines in these individuals (Paxton, et al.). However, another study reported that serum chemokine levels in patients that are infected by HIV-1 (McKenzie, et al).
The first molecular analysis of Δ32 activity utilized Δ32-like molecules to show that they interact with wild type CCR5, forming heterodimers that are retained in the endoplasmic reticulum resulting in reduced cell surface expression (Benkirane, et al.). These findings suggested that CCR5/Δ32 heterodimerization is a molecular mechanism for slower progression to AIDS in individuals with a heterozygote genotype.
There is evidence for reduced ability of CXCR4-using HIV-1 isolates to infect PBMCs isolated from -/- and +/- individuals (Wu, et al.). Recent work by Xiao, et al. demonstrated resistance to 9 primary HIV-1 R5X4 CXCR4-using Xiao, et al. isolates by lymphocytes from one exposed/uninfected -/- homozygote. Conner, et al. demonstrated that although PBMCs from EU2 and EU3 (homozygous for Δ32) were susceptible to infection by X4 isolates, a higher innoculum was needed to establish infection (Connor, et al.). Wu, et al. examined EU2 and EU3 samples used by Conner, et al. and demonstrated that the percentage of CXCR4+ cells in T cell blasts from these two individuals did not correlate with the infectibility by the X4 isolate. The primary T cells of two -/- homozygote showed very poor X4 infectivity compared to the +/+ wild type PBMCs.
Treatment of HIV infection has been revolutionized by the development of potent inhibitors of critical viral enzymes, particularly the HIV-1 reverse transcriptase and protease (Richman, D.D. (2001) Nature (London) 410:995-1001). Appropriate combinations of such drugs (referred to as highly active antiretroviral therapy or HAART) markedly suppress viral replication in most treated persons, leading to significant restoration of immune -system function. In the developed world, HAART is responsible for dramatic reductions in HIV-associated morbidity and mortality. However, the quest for improved therapies continues, because of problems that seriously limit the current HAART regimens, including toxic side effects, viral persistence, and difficulties in adhering to treatment, high cost, and the emergence of drug-resistant escape variants. The resistance problem is particularly challenging because of the extraordinarily high HIV-1 mutation rate, and the ability of viral variants harboring resistance mutations in both reverse transcriptase and protease to continue replicating in vivo. The viral mutability provides a rationale for developing alternate treatments. The cellular receptors involved in HIV-1 entry are receiving special attention, with numerous candidate inhibitors at various stages of clinical development (Eckert, D. M. &Kim, P.S. (2001) Annu. Rev. Biochem. 70:777- 810).
The Δ32 approach can therefore be useful to develop new methods and treatments of HIV since the Δ32 protein is naturally occurring and is expressed in people who resist HIV infection. Such a Δ32-based treatment does not have the side effects found in the art HAART treatment of HIV-1 infection since it is aimed at decreasing the co-receptor density in a manner that is targeting both CCR5 and CXCR4, the major molecules that are responsible for disease transmission and progression, respectively. Furthermore, individuals expressing the Δ32 protein are healthy and do not show any immunological disorders.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a method of protecting individuals from contracting HIV by administering either a vector or a vaccine containing a sequence encoding the Δ32 mutant protein containing the 31 frame shift amino acids. Also provided is a method of decreasing the amount of HIV coreceptors present on the cell surface by administering a compound having a sequence encoding the Δ32 mutation in a pharmaceutically acceptable carrier. Also provided is a compound for decreasing the amount of HIV co-receptors present in the cell surface, the compound having a sequence encoding the Δ32 mutant protein including the frame shift amino acids in a pharmaceutically acceptable carrier. A vector containing a sequence encoding the Δ32 mutant protein is also provided. An assay for testing the efficacy of HIV treatment is also provided, the assay includes a detector for detecting the presence of the Δ32 mutant protein in cells.
DESCRIPTION OF THE DRAWINGS
Other advantages of the present invention are readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:
Figure 1 is a diagram showing the membrane orientation model of the 7 TM domain chemokine receptor protein;
Figure 2 is a model showing the predicted amino acid structure of the Δ32 mutant protein;
Figures 3 A and B are graphs showing the fusion specificities of human cells expressing an adenovirus encoded CCR5 (Figure 3A) or a vaccine virus encoded CXCR4 (Figure 3B);
Figures 4A-C are flow cytometry analyses of cell surface CCR5 in a presence or absence of Δ32;
Figures 5 A and B are graphs showing the specific down modulation of CXCR4;
Figures 6 A and B are graphs showing that the cells co-expressing Δ32 and either CCR5 or CXCR4 are resistant to R5 and X4 fusion; Figures 7 A and B are graphs showing the specificity of Δ32 induced inhibition of HIV-1 Env-mediated cell fusion;
Figures 8 A and B are graphs showing the cell specificity of the Δ32 affect; Figures 9 A and B show PHA plus IL-2 activation upregulates CXCR4 and CCR5; Figures 10 A and B are histograms representing staining of uninfected PMBC cells;
Figures 1 1 A and B are graphs showing the affect of Δ32 on Env-mediated cell fusion and human PMBCs;
Figures 12 A-C are graphs showing PMBCs from individuals with known CCR5 genotype;
Figures 13 A and B are graphs showing the infection kinetics of -/- and +/+ PMBCs with HIV-1 IIIB(X4) and Ba-L(R5); Figures 14 A-C are photographs showing immunoblot analysis of Δ32 and CCR5 proteins expressed in infected 293 cells;
Figures 15 A and B are photographs showing the immunodetection of native Δ32 protein expressed in unstimulated PMBCs of three -/- homozygous individuals; Figures 16 A-C are photographs showing the expression analysis of Δ32 mRNA in -/- PMBCs (Figure 16A) and recombinant 85 infected cells (Figure 16B) by RT-PCR;
Figure 17 is a graph showing the effect of recombinant Δ32 protein in X4 infection; Figures 18 A and B are graphs showing the Phytohemagglutinin-A + IL2- activated Ficoll purified human PBMCs which were infected with either vLA-1 (Ad5/CCR5) or vLA-2(Ad5/Δ32) at 3pfu per cell for each virus for two days that were then infected with either Ba-L(R5) or IIIB(X4);
Figures 19 A and B are hypothetical models of the Δ32 affect; and DETAILED DESCRIPTION OF THE INVENTION
Generally, the present invention provides a method and composition for treating and vaccinating against HIV. The composition of the present invention can be used either as a treatment for an individual who has already contracted HIV or as a vaccine to prevent the transmission of HIV. Accordingly, the composition must be administered for a sufficient period of time or at a sufficient concentration to obtain the desired effect in the individual to whom the composition has been administered.
The composition of the present invention includes a sequence encoding the Δ 32 mutant protein and analogues and homologues thereof and a pharmaceutically acceptable carrier. The composition can be used to create a vaccine or as a gene therapy.
The composition of the present invention can be administered in any manner known to those of skill in the art. Preferably, the composition is administered either orally or intramuscularly. An adenovirus vector encoding the Δ32 mutant protein can be used as a vaccine which is preferably administered orally. An acceptable vaccine that uses the same vector used to vaccinate against smallpox can be used to vaccinate individuals.
By "vaccine" as used herein, the term is intended to include, but is not limited to a treatment which prevents HIV infection in individuals who have received the treatment thereby making the individual immune against HIV. A recombinant vaccinia virus encoding the Δ32 mutant protein has been constructed and used to infect human peripheral blood lymphocytes. Lymphocytes expressing the encoded Δ32 mutant protein showed resistance to HIV-1 entry and infection. In a further alternative, following the initial administration of the vaccine itself, a later administration of the vaccine can be given every month to maintain sufficient expression of the encoded Δ32 mutant protein. The clinical condition of the individual patient, the site and method of administration, scheduling of administration, and other factors known to medical practitioners can be taken into consideration.
The "effective amount" for purposes herein is thus determined by such considerations as are known in the art of vaccination wherein it must be effective to provide measurable anti-virus titer in persons given the vaccine, and, in a preferred embodiment, persons who are non-responsive to a standard anti-retroviral therapy. To determine if a person who was non-responsive to prior art treatments and who has been immunized with the present invention is now successfully immunized, titers can be determined, as well as proliferative assays in response to viral antigen can be run as are well known in the art.
The desired effect of the treatment is to either prevent the transmission of HIV or to prevent the progression of HIV infection in seropositive individuals. The composition accomplishes these effects by preventing effectively the expression of coreceptors (CCR5 and CXCR4) present on the cell surface responsible for transmission and progression.
By gene therapy as used herein refers to the transfer of genetic material (e.g DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype. The genetic material of interest encodes a product (e.g. the Δ32 mutant protein) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value. Alternatively, the genetic material of interest encodes a suicide gene. For a review see, in general, the text "Gene Therapy" (Advances in Pharmacology 40, Academic Press, 1997).
Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy. In ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to express the transfected genetic material in situ.
In in vivo gene therapy, target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ that is within the recipient. In an alternative embodiment, if the host gene is defective, the gene is repaired in situ (Culver, 1998). These genetically altered cells have been shown to express the transfected genetic material in situ.
The gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell. The expression vehicle can include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR of the gene can be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore as used herein the expression vehicle can, as needed, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred and only include the specific amino acid coding region. The expression vehicle can include a promotor for controlling transcription of the heterologous material and can be either a constitutive or inducible promotor to allow selective transcription. Enhancers that can be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non- translated DNA sequence, which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. The expression vehicle can also include a selection gene as described herein below.
Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Ml (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Ml (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see United States patent 4,866,042 for vectors involving the central nervous system and also United States patents 5,464,764 and 5,487,992 for positive-negative selection methods.
Introduction of nucleic acids by infection offers several advantages over the other listed methods. Higher efficiency can be obtained due to their infectious nature.
Moreover, viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.
A specific example of DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector Adenop53TK. This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences. This vector can be used to infect cells that have an adenovirus receptor, which includes most cancers of epithelial origin as well as others. This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vitro or ex vivo culture of cells, a tissue or a human subject.
Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy. Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus. An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation does not occur.
Features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are < \ specific for the desired -cell type.
In addition, recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. The vector to be used in the methods of the invention depends on desired cell type to be targeted and is known to those skilled in the art. For example, if breast cancer is to be treated then a vector specific for such epithelial cells can be used. Likewise, if diseases or pathological conditions of the hematopoietic system are to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, can be used. A recombinant adeno-associated (AAV) viral vector encoding the Δ32 mutant protein can be constructed and used to infect hematopoietic stem cells. AAV vectors are potential gene delivery viral vectors that have the advantage of being non-pathogenic to human cells. Such vectors can provide a safe delivery and integration of the Δ32 gene into hematopoietic progenitor cells. When transplanted into the patient, these cells can give rise into blood lymphocytes that express the protective Δ32 mutant protein. The cells expressing the Δ32 mutant protein are immune to infection by a wide range of HIV-1 strains including X4, R5, and X4R5.
Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection. In the former case, the genome of the virus is modified so that it maintains all the necessary genes, regulatory ' I sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles, which are capable of undergoing further rounds of infection. The vector's genome is also engineered to encode and express the desired recombinant gene. In the case of non-infectious viral vectors, the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed do not contain a genome and therefore cannot proceed through subsequent rounds of infection. The specific type of vector depends upon the intended application. The actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.
The recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neurodegenerative diseases. Following injection, the viral vectors circulate until they recognize host cells with the appropriate target specificity for infection.
An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid. Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal. Such non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like. Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art.
The compound of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
It is noted that humans are treated generally longer than the mice or other experimental animals exemplified herein which treatment has a length proportional to the length of the disease process and drug effectiveness. The doses can be single doses or multiple doses over a period of several days, but single doses are preferred.
The doses can be single doses or multiple doses over a period of several days. The treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.
The Δ32 mutation is also an effective therapeutic target for the treatment of HIV-1 infection. This target can be used for developing further compositions for treating HIV infection. For example, new compounds can be developed which target both CCR5 and CXCR4 coreceptors and thereby inhibiting HIV transmission and progression. Further, additional gene therapy products, such as new vectors can be developed which target the major HIV coreceptors. Studies analyzing the molecular effect of this mutation have largely relied on the introduction of the Δ32 DNA into cells by transfection. This method results in an inefficient delivery of the Δ32 into the cells and results in expression of Δ32 protein in very few cells in a sample monolayer. Applicants have developed adenovirus and vaccinia virus vectors that deliver the Δ32 DNA efficiently into every cell in the monolayer. As a result, 100% of the cells transduced with the Δ32 vector express this protein (Δ32 protein).
The discovery of the novel Δ32 protein activity is directly related to AIDS pathogenesis. The results indicate that Δ32 downmodulates CXCR4, the coreceptor used by HIV-1 at late stages of the disease, leading to AIDS. Undestanding the mechanisms of this novel activity by the Δ32 protein and its protective phenotype against CXCR4-using HIV-1 strains leads to the development of drugs that mimic the Δ32 action. Such drugs are extremely helpful for prevention of disease progression and viral transmission.
A growing body of evidence shows that Δ32 heterozygosity (individuals carrying one copy of Δ32) is associated with reduced risk to some complications of the disease. For example, studies analyzing the effect of Δ32 genotypes indicated reduced prevalence of Δ32 mutation in those who do develop AIDS dementia complex (ADC). Others reported an association of Δ32 mutation with protection against HIV-associated lymphoma. Recent reports described that carriers of the Δ32 mutation respond better to highly active antiretroviral therapy (HAART) treatment compared to wild type controls. Finally, CD8+ T cells purified from Δ32 carriers showed increased ability to kill virally infected cells. All the above studies point to the potential use of the Δ32 effect in other disease conditions.
The ability of humans to resist infection by viral, bacterial, and parasitic pathogens can be influenced by host genetic factors. Human immunodeficiency virus type 1 (HIV-1) employs CD4 and a coreceptor, principally the CCR5 and/or CXCR4 chemokine receptors, for entry into host cells. The central role of CCR5 in HIV-1 transmission and pathogenesis has been highlighted by the epidemiological and genetic identification of powerful disease modifying effects of the naturally occurring CCR Δ32 allele, a 32 base pair deletion encoding a truncated and non-cell surface expressed version of the receptor.
Relative to the general population, CCR Δ32 homozygotes are rarely found among HIV-1 infected individuals. In addition, HIV-1 infected CCR Δ32 heterozygotes progress more slowly to AIDS than individuals lacking this allele. Previous studies have indicated that Δ32 protein binds to wild-type CCR5 inside the cell and can retard the transport of functional CCR5 to the cell surface. However, this mechanism did not explain why CXCR4 only rarely could compensate for the CCR5 deficiency and allow X4 or dual tropic (R5X4) virus infection. Instead, the present invention shows that the Δ32 protein down-regulates the major coreceptors resulting in an unfavorable of the molecules involved in membrane fusion. Consistent with this, it has been found that recombinant Δ32 protein expression, by recombinant adenovirus in human cell lines or in human peripheral blood mononuclear cells can dramatically and specifically reduce the expression of endogenous CXCR4 at the cell surface resulting in the inhibition of virus entry and infection by CXCR4-using X4 HIV-1 isolates. This effect was not observed in cells infected with a CCR5 encoding or a measles virus hemaglutinin (MVHA)-encoding recombinant adenovirus. Using Δ32-specific antibodies, Δ32 is expressed in Δ32/Δ 32 PBMCs. PBMCs from Δ32/Δ32 individuals express lower surface levels of CXCR4 in comparison to wild-type CCR5 PBMCs. Further, lower X4 fusion/infection activity was correlated in the Δ32/Δ32 cells. To analyze the role of Δ32 mutation in resistance to HIV-1, replication- defective adenovirus type 5 (Ad5) vectors were constructed encoding either CCR5 or Δ32 proteins. The Ad5 gene delivery system results in efficient delivery of the gene of interest to most cells in a monolayer resulting in sufficient expression levels to allow detailed analysis and characterization of the Δ32 protein and examination of its potential role in the protective phenotype. One mechanism of genetic resistance to HIV-1 is caused by the unique activity of the Δ32 protein that results in an unfavorable stoichiometry of the surface proteins involved in HIV-1 entry.
Analysis of Δ32 protein in -/- PBMC samples indicated reduced CXCR4 levels in -/- samples compared to +/+ PBMCs. The reduced surface expression correlated with reduced cell fusion and HIV infectivity (Section C9). Moreover, Δ32 expression in human PBMCs or in MAGI-CCR5 cells reduced their susceptibility to R5 and X4 infections. Recent studies by Lee, et al. reported coreceptor competition for association with CD4 and demonstrated that introduction of CCR5 by a recombinant vaccinia vector caused reduction in X4 fusion. The data of the present invention are not conflicting with those recently published by Lee, et al. since a replication- defective adenovirus was used to express CCR5 while Lee, et al. used a replication- competent vaccinia virus that expresses much higher levels of CCR5. In the present system, a slight reduction in X4 fusion upon CCR5 expression was detected, but was not comparable to the effect obtained with Δ32. It is possible that the vaccinia virus infection caused those effects in certain CD4+cell lines. Furthermore, the analysis utilized a quantitative method to assay for cell fusion while Lee, et al. measured the effect by scoring syncytia. Δ32 interacts specifically with the major coreceptors CXCR4 and CCR5 and impairs the formation of coreceptor complexes and CD4-coreceptor interaction resulting in an imbalance of the optimal stoichiometry of the molecules involved in the process of membrane fusion. The experimental approach described herein enables one to characterize the Δ32 protein at the molecular level and determine its contribution to resistance to HIV-1 infection. The observed effect of Δ32 on both CCR5 and CXCR4 implicates a common interaction site for the principal coreceptors on Δ32.
Biological activity of Δ32 protein encoded by a recombinant vaccinia virus (vZV-1) was also studied. This can also be in the form of an attenuated vaccinia virus that is used for smallpox vaccination. A recombinant vaccinia virus that expresses the Δ-32 protein with expression that results in cells that are resistant to HIV-1 entry has also been established. The results of the experiment are shown in Figure XY. Cells (MAGI-CCR5) were infected with recombinant vaccinia viruses encoding the proteins indicated at the X-axis. These cells were challenged with cells expressing the two major prototypic HIV-1 envelope glycoproteins (LAV or Ba-L). The extent of cell fusion was determined by measuring the production of β- galactosidase. The results show that cells expressing Δ32 are resistant to HIV-1 fusion. Two controls were included in this experiment. The first control is the use of cells infected with the vaccinia vector (WR), the vector used to generate the Δ 32/vaccinia recombinant virus. The second control is the use of cells infected with a vaccinia recombinant encoding CCR5. The two controls showed no resistance to HIV-1 fusion as demonstrated in the figure.
The absence of Δ32 protein from an infected Δ32/Δ32 homozygous individual was also investigated. Delta-32 RNA and protein expression in (-/-) PBMCs was also examined. RNA expression of Δ32 was verified by RT-PCR analysis. The immunoblot analysis revealed a protein band that corresponds to the same molecular weight band seen with recombinant protein analysis. These protein bands were not obtained with the preimmune serum. The identity of the band that appears above the 34 Kd marker band is not known at present, however, since it is expressed in normal CCR5 individuals, it could represent a cross-reactive cellular protein. The analysis also revealed that the Δ32 protein band detected in the protected (-/-) individuals was absent in an infected (-/-) individual suggesting a critical role for the Δ32 protein in resistance to HIV-1.
The recombinant vaccinia virus encoding the Δ32 protein can be used as a live attenuated vaccine to deliver the Δ32 protein into susceptible cells. The mechanism of protection obtained with this recombinant virus will be investigated in a mouse model that expresses human CXCR4, CCR5 and CD4. The mode of administration can be intramuscular or intravenous. The C-terminal region of the Δ32 protein created novel epitopes that are not native to people who do not express Δ32 protein. Therefore, antibody generation to those novel epitopes in Δ32 occur in vaccinated mice. These antibodies can be examined for their ability to neutralize HIV-1 infection.
Adeno-associated viral vectors (AAV) vectors are also potential vectors for gene therapy (Zhou, et al., Gene Therapy, 3:223-229 (1996)). These vectors also contain the herpes virus TK promoter-driven neomycin gene for selection. AAV- based vectors have the advantage of being non-pathogenic and the fact that they integrate into the host chromosome by a site-specific manner. Therefore, they do not appear to lead to insertional mutations. HIV-based lentivirus vectors can also be utilized. Recombinant HIV-1 vector expressing the Δ32 protein can be constructed and used to treat HIV-1 infection in cultured peripheral blood lymphocytes that are chronically infected with HIV-1. The idea is that the recombinant Δ32/HIV integrates into the host chromosome at the same sites that contain the wild type genomic copies of HIV-1. Recombination results in the insertion of the Δ32 gene into the host chromosome thereby providing continuous expression of the Δ32 protein. This approach can be very beneficial for those patients who do not respond to HAART and are therefore left to their fate and to the virus controlling their immune systems. The idea of generating HIV-based vectors to trap HIV-1 virus has been previously suggested (Endres, et al., Science, 278:1462-1464 1997). This approach offers a method for delivering the Δ32 protein directly to HIV-infected cells in vivo and provides an additional treatment strategy in conjunction with existing HAART therapy.
Therefore, detailed analysis of the mechanism of Δ32-Coreceptor interaction leads to the design of therapeutic strategies aimed at targeting the major coreceptors. These studies provide insight into the molecular nature of genetic resistance to HIV-1 and have important implications for understanding coreceptor structure-function and mechanism of virus entry.
EXAMPLES
GENERAL METHODS:
General methods in molecular biology: Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), and in Ausubel, et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989) and in Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), and in Watson, et al., Recombinant DNA, Scientific American Books, New York and in Birren, et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998) and methodology as set forth in United States Patents 4,666,828; 4,683,202; 4,801 ,531 ; 5,192,659 and 5,272,057 and incorporated herein by reference. Polymerase chain reaction (PCR) was carried out generally as in PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, CA (1990). In-situ (In-cell) PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences (Testoni et al, Blood, 87:3822 (1996)).
Recombinant Protein Purification
Marshak, et al, "Strategies for Protein Purification and Characterization. A laboratory course manual." CSHL Press, 1996.
Example 1 : In order to study the role of Δ32 in genetic resistance to HIV-1 infection, a vector system is needed that can reproducibly deliver the Δ32 transcription unit into most cells in a monolayer that result in sufficient quantities of recombinant Δ32 in a variety of cell types. The immunological reagents must also be developed that enable one to specifically detect native and recombinant Δ32 proteins. A number of cDNA clones have been obtained encoding several CC and CXC chemokine receptors. Most of these receptors were subcloned under the control of the synthetic early/late promoter of vaccinia virus to produce higher levels of expression of the chemokine receptor protein. The original cDNA clones were all under the T7 promoter, and are used when lower levels expression of the protein is desired.
A number of recombinant vaccinia viruses made by Chris Broder were obtained from the laboratory of Ed Berger, N1AID.NIH. These recombinants are used to prepare cells expressing a variety of HIV-1 envelope glycoproteins (Envs) derived from different X4, R5, or R5/X4 isolates.
The immuno-detection of recombinant proteins is critical to verify expression, localization, and quantification of the molecules being tested. Polyclonal antibodies against synthetic peptides corresponding to the amino termini of CXCR4 (Feng, et al.) and CCR5 (Alkhatib, et al.) have been described and are available for use in the laboratory. It was shown that these antibodies detect recombinant CXCR4 expressed by using the vaccinia virus vectors, and endogenous CXCR4 made in peripheral blood macrophages and lymphocytes. The anti-CXCR4 peptide antibodies, however, do not detect surface CXCR4. For surface detection experiments monoclonal antibodies are used that have been obtained commercially. It was previously shown that the anti-CCR5 peptide antibodies generated are very efficient in detecting recombinant (Alkhatib, et al.). Commercial antibodies to the various chemokine receptors are now available. Synthetic peptides corresponding to the amino termini of CCR4, CCR8, CCR1 , and STRL33 can be ordered to generate monospecific antisera in rabbits.
During the last year a number of recombinant viruses were constructed that encode some of the HIV coreceptors that are currently being studied. Most importantly, a recombinant adenovirus encoding either wild type CCR5 or the deletion mutant Δ32 protein, referred to as Δ32 has been constructed. The entire cDNA fragments encoding either Δ32 or CCR5 were individually
, cloned into the adenovirus plasmid ' under control of cytomegalovirus (CMV) promoter. The method for generating recombinant adenoviruses encoding either Δ
32 or CCR5 is detailed below. Dot blot analysis of virion-derived Ad5/Δ32 or
Ad5/CCR5 DNA was used to isolate positive recombinant viral plaques on 293 cell monolayers. Southern blot analysis was subsequently performed to confirm the predicted structure of recombinant viruses without deletions or rearrangements, even after three rounds of plaque purification and propagation. The recombinant viruses consistently grew to titers ranging between 7x108 to 1x109 plaque forming units (PFU)/ml in 293 cells.
Infection with wild type Ad5, Ad5/MVHA, or AdδMVPC, using two different recombinant Ad5 viruses encoding either measles virus (MV) haemaglutinin (Alkhatib, et al.) or MV phosphoprotein Alkhatib, et al.) were used as negative controls for all subsequent experiments to measure the effect of recombinant Δ32 protein on cell surface expression of the major coreceptors and their fusion activities. Wild type Ad5 is a replication competent virus that does not contain DNA sequences that are specific for Δ32 or any chemokine receptor. Expression of MVHA or MVPC by Ad5 was verified by surface staining using MV-specific polyclonal antibodies as previously described (Alkhatib, et al.).
In order to isolate recombinant plaques, dot blot analysis was first used to identify hybrid viruses containing either CCR5 or Δ32 DNA sequences. Cell lysates from different plaques, amplified in 293 monolayers, were prepared by alkaline lysis and blotting onto nylon membranes (Millipore). After washing and blocking, the nucleic acids were UV crosslinked (Stratagene UV crosslinker). Positive plaques were identified by probing the membrane with a CCR5-specific DNA fragment that corresponds to the DNA sequence of Δ32 and CCR5 DNA.
The experimental approach involves the analysis of fusion between two distinct cell populations, one expressing CD4 (endogenous or encoded by a recombinant virus) and the other expressing the indicated HIV-1 envelope glycoprotein encoded by a recombinant vaccinia virus. Cell fusion is scored by a reporter gene activation assay in which the cytoplasm of one cell population expressing vaccinia virus-encoded T7 RNA polymerase and the cytoplasm of the other expressing lac Z gene linked to the T7 promoter; cell fusion activates β- Galactosidase.
The advantages of preparing target cells (cells expressing the coreceptor) using the Ad5 expression system include:
1) The Ad5 cytopathic effect is much less severe than vaccinia, 2) The cells are healthier and easier to manipulate; 3) The recombinant Ad5 viruses are efficient high-level expressing vectors that result in infection of all cells in a monolayer; 4) The G-protein signaling pathway is not impaired in Ad5-infected cells (Braciak, et al.) compared to vaccinia virus infected cells; 5) The recombinant Ad5 viruses are replication-defective in cells other than 293 and can be useful to analyze without the cytopathic effect associated with the wild type virus; and 6) The recombinant Ad5 viruses provide a much better signal to background ratio.
Co-expression of proteins is accomplished with the Ad5 vector system by infecting cells with two different recombinant viruses at the same time. This is a well established method for expressing two different proteins in a variety of cell types due to the broad host specificity of Ad5 for mammalian cells (Alkhatib, et al.). The basic features of the fusion assay were developed by using the HIV-1 Env-CD4 interaction of two different populations of cells, one expressing CD4 and the other expressing the HIV-1 Env (Nussbaum, et al., Alkhatib, et al.). The rationale for using an adenovirus expression system is that cells infected with this vector can, unlike vaccinia infected cells, respond to calcium flux assays making it possible to analyze the response of infected cells to different chemokines. Moreover, the recombinant Ad5 viruses used are replication-defective and do not cause severe cytopathic effect in a wide range of mammalian cells including primary cells. In the vaccinia-based cell fusion assay, CCR5 functions as a fusion coreceptor for Envs from several R5-tropic HIV-1 isolates, as well as the R5/X4 strain 89.6 (Alkhatib, et al.). In order to examine whether CCR5 expressed by the recombinant adenovirus is functional with the same fusion specificity, human 293 cells were used as the target cell population after transfection with pCDNA3/CD4 and coinfection with Ad5T7β-gal (lacZ under T7, from Frank Graham and vLA-1 (CCR5). Hela cells coinfected with vTF7-3 (T7 RNA polymerase) and one of the HIV-1 Envs as effector cells were used. After mixing the effector and target cell populations and incubation at 37°C for 2.5 hours, fusion specificity of HIV-1 Envs was measured by β-gal production in a colorimetric lysate assay. The results indicate that 293 cells expressing CCR5 showed the expected R5-specific coreceptor activity and fused with cells expressing the R5 Envs (Figure 3A). In contrast, 293 cells expressing endogenous as well as vaccinia virus encoded CXCR4 did not show R5-specific fusion (Figure 3B). This demonstrates that CCR5 protein encoded by the recombinant adenovirus is a biologically functional protein in terms of its well-documented coreceptor activity. The observed X4 fusion activity with LAV Env in CCR5-expressing cells (Figure 3A) is due to endogenous expression of CXCR4 by 293 cells. Figure 3 shows the. fusion specificities of human 293 cells expressing either adenovirus-encoded CCR5 Figure 3(A) or vaccinia virus encoded CXCR4 Figure 3(B). Human 293 cells expressing Ad5-encoded CCR5 or vaccinia-encoded CXCR4 were challenged with effector cells expressing the indicated HIV-1 Envs. Fusion is scored by the amount of β-gal accumulated. The results of this analysis were confirmed at least three times. In order to examine whether the Ad5-encoded Δ32 protein is a biologically active protein, cells coexpressing Δ32 and CCR5 were analyzed for cell surface expression of CCR5 (Figures 4A-C). Analysis of cell surface expression of CCR5 was performed in the absence or presence of recombinant Δ32 protein. Recombinant adenoviruses encoding either CCR5 or Δ32 proteins were used to express these proteins in 293 cells.
Monoclonal antibodies to CCR5 (PDL) were used to detect coreceptor density at the cell surface of infected cells. Surface levels of CCR5 were significantly reduced in the presence of recombinant Δ32 protein (Figure 4), consistent with previous published observations that examined the effect of Δ32 on cell surface expression of CCR5 (Benkirane, et al., Wu, et al., Paxton, et al.).
To examine whether the downmodulation induced by Δ32 is specific for CCR5 and CXCR4, a human CCR5/293 cell line and another CXCR2/293 cell line (provided by Phil Murphy, NIH) were used to analyze the effect of Δ32 on CXCR4. Human CCR5/293 cells were infected with VLA-2 Δ32), vLA-1 (CCR5), AdδMVHA (MVHA), AdδMVPC (MVPC), or Ad5 and stained with Mabs against CXCR4. The results of this analysis demonstrate that cells expressing Δ32 but not CCR5, MVHA, or MVPC showed downmodulation of CXCR4 (Figure 5A). Expression of MVHA and MVPC was verified by antibody detection as previously described (Alkhatib, et al.). It was also examined as to whether Δ32 expression in a human CXCR2/293 cell line has an effect on surface levels of CXCR2. CXCR2 is a CXC chemokine receptor that shares 35% homology with CXCR4.
Expression of Δ32 in CXCR2/293 cells was accomplished by infection with vLA-2 at increasing moi's. As negative controls, was examined CXCR4 staining in this cell line infected with AdδMVHA or vLA-1 (CCR5). The staining results (Figure 5B) indicate that CXCR4 was specifically downmodulated (Figure 6B, Δ/CXCR4) with no significant effect on CXCR2 expression (Figure 5B, Δ/CXCR2).
Additionally, CXCR2 or CXCR4 surface levels were not significantly altered in cells expressing either CCR5 or MVHA - (Figure 6B, CCR5/CXCR2 and MVHA/CXCR4 line graphs) indicating the specificity of Δ32 downmodulation effect. In other experiments, the effect of Δ32 expression on the epidermal growth factor receptor (EGFR), a glycoprotein expressed on most cells including 293 cells and on CCR2 in a human CCR2/293 cell line (provided by Phil Murphy, NIH) were examined.
The same population of 293 cells infected with vLA-2 Δ32) was used to examine EGFR and CCR2 expression as well as endogenous CXCR4. Cell surface EGFR or CCR2 was not significantly affected as a result of Δ32 expression. Figure 4 shows the flow cytometry analysis of cell surface CCR5 in the presence or absence of Δ32. Cells (293 cells) expressing CCR5 and/or Δ32 were treated with monoclonal antibodies and detected by indirect staining using fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G. The values shown at the right corner of each histogram represent mean fluorescence intensity.
Figure 5 shows the specific downmodulation of CXCR4. Figure 5A shows human 293 cells which were infected at 10 pfu/cell with the different Ad5 viruses encoding wild type adenovirus proteins (Ad5), measles virus hemaglutinin (MVHA), measles C& phosphoproteins (MVPC), CCR5, or Δ32 and stained for surface CXCR4. Reduction in surface CXCR4 was only observed with cells expressing Δ32 but not other proteins such as CCR5, MVHA, or MVPC.
Figure 5B shows Δ32-specific downmodulation of CXCR4 in a CXCR2/293 cell line. Cells were infected at increasing moi's with either vLA-1 (CCR5/Ad5), Ad5/MVHA, or vLA-2 (Ad5/Δ32), then stained with monoclonal antibodies to CXCR4 or CXCR2 (R&D). Note that Δ32 expression resulted in reduced surface staining of CXCR4 (Δ/CXCR4) but had no significant effect on CXCR2 surface expression (Δ /CXCR2). Expression of MVHA, or CCR5 by the adenovirus system had no downmodulation effect on cell surface expression of CXCR4 or CXCR2.
After confirming the ability of Δ32 to downmodulate both CXCR4 and CCR5 its effect on Env-mediated cell fusion activity was examined. A human 293/CD4 Cell line was generated and cells were infected with either vLA-1 (CCR5), vLA-2 Δ32), Ad5, or coinfected with vLA-1+vLA-2 (CCR5+Δ32) then infected with Ad5Pol3 (T7 RNA polymerase). Separate populations of Hela cells were infected with vCB-21 R and either vCB-16 (Unc), . vCB-41 (LAV), or vCB-39 (ADA). Fusion activity was scored by the amount of β-galactosidase produced. The results of this analysis indicate that Δ32 impaired the ability of cells expressing CCR5 and CXCR4 to fuse with R5 or X4 Envs (Figure 6). Previous studies showed that coexpression of wild type CCR5 and its truncation mutants, one of which resembled Δ32 in lacking the last three TM domains, dramatically reduced the ability of cells to support infection by HIV-1 ADA Env but had no effect on infection by NL4-3, a X4-tropic isolate (Benkirane, et al.).
Figure 6 shows cells coexpressing Δ32 and either CCR5 or CXCR4 are resistant to R5 and X4 fusion. Human 293 cells expressing CCR5 and CXCR4 (endogenous), or coexpressing CCR5+Δ32 or Δ32 were challenged with effector Hela cells expressing the indicated HIV-1 Envs. Cell fusion was scored by β-gal production. This analysis is representative of at least three different experiments.
The studies by Benkirane et al did not use the complete coding region of Δ32 ORF in their infection assay. Instead, they used PCR fragments, amplified using CCR5 DNA, as a template to construct a mutant that, similar to Δ32, lacks the last three transmembrane domains. Therefore, their Δ32 construct does not contain the Δ32 DNA fragment encoding the 31 amino acid residues. All the experiments were performed using the authentic Δ32-encoding DNA fragment isolated from an individual homozygous for the CCR5 mutant allele (Zimmerman, et al.) encoding the complete amino acid sequence of the frame shift mutation that contains 31 amino acid residues not encoded by wild type CCR5. The DNA sequence and identity of the Δ32 clone was confirmed by direct sequencing of the isolated DNA fragment before and after it was cloned into expression vectors (Zimmerman, et al.). Recently, Lee, et al. analyzed the effect of the relative density of surface CD4 on human T cells and the ability of CCR5 to reduce CXCR4-mediated cell fusion. This is not concordant with the result of these experiments probably because of the different vector system used by Lee, et al. They used vaccinia virus vector to introduce very high levels of CCR5 in certain T cell lines, while the present experiments used the replication-defective adenovirus system, which expresses 50X less CCR5 than vaccinia. Moreover, the method of detection could contribute to the different results. X4 fusion was measured by a quantitative method while Lee, et al. used microscopic counting of syncytia. Specific inhibition of HIV-1 Env-mediated cell fusion bv Δ32: HIV-1 VS human T cell- leukemia virus type 1 (HTLV-1) Env-mediated fusion.
In order to determine the specificity of Δ32-induced inhibition of HIV-1 fusion, the effect of recombinant Δ32 expression on HIV-1 and HTLV-1 Env-mediated cell fusion were compared.
Recombinant vaccinia viruses encoding HTLV-1 Env or its transmembrane gp46 subunit were obtained from Dr. H. Shida. gp46 expression was used as a negative control for the specific HTLV-1 Env-mediated cell fusion. This choice was based on the fact that HTLV-1 is a human retrovirus that has a broad range specificity for mammalian cell lines (Sutton, et al.). Target cells expressing Δ32 showed specific reduction in X4 fusion when challenged with the X4 LAV Env. In contrast, the same target cells challenged with HTLV-1 Env-expressing cells showed comparable levels of cell fusion observed with control cells expressing CCR5, MVHA or T7 RNA polymerase (pol3) (Figure 7).
More specifically, Figure 7 shows the specificity of Δ32-induced inhibition of HIV-1 Env-mediated cell fusion. Target cells (293 or MAGI- CCR5) were coinfected with Ad5pol3 (T7RNA polymerase) and either vLA-2 (pol3+Δ32) vLA 1 (pol3+CCR5).
The effect of Δ32 on Env fusion was analyzed using three different cell lines. Figure 8 shows results obtained by the quantitative assay of β-gal in detergent cell lysates. With 293 cells and MAGI-CCR5 cell line, X4 and R5 fusion showed dramatic reduction as a result of Δ32 expression, whereas no significant effect on X4 fusion was observed with HL60. Control cells expressing recombinant CCR5 (coinfected with vLA-1+pol3) did not show significant reduction in X4 fusion. Interestingly, when HL60 cells were induced to differentiate into macrophages by RA treatment, the differentiated cells showed the Δ32 effect (Figure 8B).
These results show that the presence of another cellular protein is required for Δ32 activity. The identity of this cellular factor is currently unknown but it raises questions about possible differences in the efficiency of Δ32 activity in different cell types.
Figure 8 shows the cell specificity of the Δ32 effect. The effect of Δ32 on X4 fusion is observed with MAGI-CCR5 and 293 cell lines but not with HL60 cell line Figure 8(A). Retinoic acid (RA) treatment of HL60 cell induces them to differentiate and become sensitive to -the Δ32 effect Figure 8(B). Cells were transfected with pCDNA3/CD4 to provide CD4 expression. All cell types were coinfected with pol3 (no Δ32) or vLA 2+pol3 (+Δ32) and challenged with Hela cells expressing X4 Env (LAV) or R5 (Ba-L) and pT7-lacZ. Cell fusion was measured by β-gal produced. The effects of cell activation on CCR5 and CXCR4 expression in PBMCs isolated from seven healthy volunteers who are HIV seronegative were examined. Cells were stimulated with PHA, PHA+IL-2, or kept unstimulated in RPMI 1640 containing antibiotics and 10% FBS. The results indicate that unstimulated PBMCs express detectable levels of CXCR4 and CD4 but not CCR5 (Figure 9A). The effect of cell activation on cell fusion activity of PBMCs with HIV-1 Envs was examined. Although unstimulated PBMCs express detectable levels of CXCR4 and CD4, they do not show significant fusion activity with X4 Envs (Figure 9B).
Stimulation with PHA+IL-2 increased CXCR4 expression and X4 fusion by at least twofold. Surface levels of CXCR4 and CCR5 are upregulated upon PHA stimulation in all seven PBMC samples examined. These results are in agreement with published data (Bleul, et al.).
Depending on the method used for PBMC activation, it is possible to get different results in HIV-1 infection assays. A recent study on the effect of CD3+CD28 stimulation of PBMCs demonstrated that CD3+CD28 stimulation produces cells that are susceptible for X4 isolates but are resistant to R5 viruses (Carroll, et al.).
Explanation for the inability of X4 HIV-1 Envs to fuse with unstimulated PBMCs, which express detectable levels of CD4 and CXCR4 is not available. A possible scenario is that CD4 and CXCR4 levels in unstimulated PBMCs may not be the optimum levels needed for cell fusion. Augmenting CXCR4 and/or CD4 expression renders these cells competent for X4 fusion.
Figure 9 shows that PHA+IL-2 activation upregulates CXCR4 and CCR5. PBMCs from seven healthy individuals were stimulated with either PHA Figure 9(A) or PHA+IL-2 and used for cell surface staining of CCR5 and CXCR4 Figure 9(A) or cell fusion assay using one PBMC sample Figure 9(B). These results show that CXCR4 levels are variable among individuals confirming published data (Lee, 1999). Interestingly, unstimulated PBMCs respond well in signaling assays using SDF-1 for receptor activation. It appears that the levels of CXCR4 required for signaling assays are different from those required for fusion/infection. The effect of recombinant Δ32 was examined in PBMCs freshly isolated from healthy donors. vLA-2 was used to express recombinant Δ32 in PHA+IL-2 stimulated PBMCs and examined cell surface levels of CXCR4, CD4 and CD44 by FACS analysis. A slight downmodulation effect of CD4 (15-20%) and no effect on CD44 surface expression was observed (Figure 10A). In contrast, appreciable downmodulation of CCR5 and CXCR4 was observed in the same PBMC population tested (Figure 10B). Ad5/CCR5 is a replication-defective recombinant virus that does not replicate in primary cells and therefore, it is not expected to result in a robust increase in the levels of CCR5. CCR5 staining of Ad5/CCR5-infected PBMCs showed an increase in CCR5 expression (from 370 to 404, Figure 10B) confirming the delivery of recombinant CCR5 in primary cells.
In other experiments using different PBMC samples significant change in CD4 surface levels as a result of Δ32 protein expression was not observed. Similar experiments were performed with Hela CD4 and MAGI-CCR5 cell lines and the results indicated that CD4 levels were not affected by expression of Δ32. In all of these experiments, significant downmodulation of the CCR5 and CXCR4 was always observed as a result of Δ32 expression. The 15 %-20% reduction in CD4 expression with the PBMC sample shown in Figure 10A was only observed with that one PBMC sample. The Δ32 effect on HIV-1 Env-mediated cell fusion with PHA+IL-2 activated
PBMC sample was analyzed by infecting with vLA-1 , vLA2, or Ad5/MVHA and vCB- 21 R (T7-lacZ). Hela cells expressing T7 RNA polymerase and the X4 LAV or Unc Envs were used as effector cells. After mixing the two cell populations and allowing the fusion reaction to incubate for two hours, detergent-treated cell lysates were used to quantitate the amount of β-gal production as a result of cell fusion. The results demonstrate the ability of recombinant Δ32 to inhibit X4 fusion in these primary cells (Figure 11 A). PBMCs expressing recombinant CCR5 (Figure 11 B) or MVHA showed no significant reduction in X4 fusion.
PBMC samples isolated from wild type +/+ and homozygous -/- individuals were used to compare cell surface expression of CXCR4. PBMCs were stimulated with PHA+IL-2 for four days then used for FACS staining, cell fusion, and HIV-1 infection assays. The results demonstrate a trend towards lower CXCR4 staining in -/- PBMCs (Figure 12A). Cell fusion activity with LAV (X4) of -/- cells is always lower th,an that observed with +/+ cells (Figure 12C). Surface expression of CD4 was variable in both +/+ and -/- PBMCs and did not seem lower in -/- compared to +/+ cells (Figure 12B). The observed CD4 variability is unlikely to influence the data presented here in terms of lower CXCR4 found in -/- individuals since it was observed in both +/+ and -/- samples.
More specifically, Figure 1 1 shows the effect of Δ32 on Env-mediated cell fusion in human PBMCs. The same PBMC samples used for FACS staining in Figure 10B were used in this cell fusion analysis. PBMCs were infected with either vLA-1 (Ad5/CCR5) or vLA-2 (Ad5/Δ32) at increasing PFUs/cell to ensure expressing recombinant protein in all cells in the monolayer. Infected PBMCs were challenged with Hela cells expressing either Unc or the X4 LAV. Unlike PBMCs expressing recombinant CCR5 Figure 1 1 (B) or MVHA, PBMCs expressing recombinant Δ32 showed a dramatic reduction in X4 fusion Figure 1 1 (A).
PBMCs were infected (105 cells) with the X4 lab adapted isolate 1MB or the R5 Ba-L and sample supernatants were collected every three days over a 15 days period. The results of this experiment demonstrate that +/+ cells infected with IIIB produced higher amounts of p24 indicating a more efficient productive infection compared to -/- cells (Figure 13). CXCR4 expression in -/- homozygotes is different from that seen in normal +/+ individuals in terms of surface density and coreceptor function.
Figure 12 depicts the results of experiments where PBMCs from individuals with known CCR5 genotype were used. Genotypes are represented by (+) for a wild-type allele and (-) for a Δ32 allele. PBMCs from -/- individuals express lower surface levels of CXCR4, show less cell fusion activity with X4 Env (LAV) and are less susceptible to X4 fusion than +/+ cells. PBMCs from -/- and +/+ individuals were stimulated with PHA+IL-2 for 4 days and used for staining Figure 12(A), cell fusion Figure 12(B), and IIIB infection. In cell fusion experiments, cells were infected with vCB-21 R (lacZ) and challenged with Hela cells expressing LAV Env and vTF7-3 (T7 RNA Polymerase). Cell fusion was scores by the amount of accumulated β-gal. As expected, Ba-L infection of +/+ PBMCs resulted in production of p24 antigen that peaked at day 6. However, productive infection with Ba-L was not observed with -/- PBMCs. One -/- sample showed a p24 peak value of 2.8ng/ml (sample#2(-/-), Figure 13, Ba-L) while another sample (#8(-/-)) showed a peak p24 value at 2.85ng/ml at day 12. It is possible that the present method of PBMCs activation (PHA+IL-2 for 4 days) resulted in upregulation of another coreceptor that can be used by Ba-L at a low efficiency. Infection in the presence of AZT showed background levels of p24 that were below 0.5ng/ml. More specifically, Figure 13 shows the infection kinetics of -/- and +/+ PBMCs with HIV-1 IIIB (X4) and Ba-L (R5). PBMCs were stimulated with PHA+IL-2 for 4 days and used in the infectivity assay. Infection was performed in a 96 well plate (10 5 cells/well). Cells were innoculated with IIIB (10 5.5 lU/ml) or Ba-L (10 4.5 lU/ml) and p24 production was measured. To obtain antisera specific for Δ32, rabbits were immunized with a peptide corresponding to the carboxy terminus of the protein (IKDSHLGAGPAAACHGHLLLGNPKNSASVSK). The carboxy terminal region was chosen because this region has no shared amino acid homology with CCR5, and antibodies to this domain are specific to the native Δ32 protein. The peptide was conjugated to KLH, and rabbits were immunized.
Immunoblot analysis was performed to verify expression of Δ32 and CCR5 proteins in 293 cells infected with vLA-2 Δ32, vLA-1 (CCR5), or wild type Ad5. A set of these samples was probed with CTC-6 monoclonal antibodies against CCR5 (Protein Design labs). This antibody detected CCR5 but not Δ32 protein (Figure 14A). When antibodies generated against the amino terminus of CCR5 were used (Alkhatib, et al.), both CCR5 and Δ32 could be detected at the correct molecular weight of the predicted amino acid sequence of each protein (Figure14 C). A Δ32 protein was detected around 30 KD between the 28 and 34 KD marker bands. The identity of the 34 KD band detected with the C-terminal antibodies (Figure 14B) is currently under investigation. The band above the 50 Kd marker band detected in both CCR5 and Δ32 expressing cells could not be CCR5 since it migrates slower (above 50 Kd) than CCR5 that migrates around the 46 Kd marker band (Figure 14 A). These experiments verify expression of recombinant CCR5 and Δ32 proteins in vLA-1 and vLA-2-infected cells. Figure 14 shows the immunoblot analysis of Δ32 and CCR5 proteins expressed in infected 293 cells. Cells were infected with vLA-1 , vLA2, or Ad5 at 10 pfu/cell for 16 hours. Cell lysates were prepared, fractionated on SDS-PAGE, and immunoblotted onto PVDF membrane (Millipore). After blocking, membranes were ) ) reacted with CTC-6 monoclonal antibodies to CCR5 (PDL), and washed. Protein bands were detected by probing with a HRP-conjugated secondary antibody and the addition of substrate. Figure 14(A) shows the monoclonal antisera detect CCR5 but not Δ32 (10% SDS-PAGE); Figure 14(B) shows the C-terminal antibodies detect Δ32 (12% SDS-PAGE) ; and Figure 14(C) shows N-terminal antibodies that detect both CCR5 and Δ32 proteins (10.5% SDS PAGE).
Δ32 protein expression in -/- PBMCs was examined using the Δ32-specific antisera. Cell lysates were prepared from unstimulated cells and analyzed the same way described for recombinant Δ32 protein described above. The immunoblot containing three -/- and three +/+ samples was probed with anti-Δ32 antibodies generated against the carboxy terminus of Δ32. The analysis revealed a protein band of different intensity for each -/- individual that corresponds to the same molecular weight band seen with recombinant protein analysis (Figure 15). Approximately, equivalent amounts of protein cell lysates were loaded in each lane. Equal gel loading was verified by staining a similar gel with commassie blue stain. These protein bands were not obtained with the preimmune serum. The identity of the band that appears above the 34 Kd marker band is not known at present, however, since it is also expressed in normal CCR5 individuals, it could represent a crossreactive cellular protein. More specifically, Figure 15 shows the immunodetection of native Δ32 protein expressed in unstimulated PBMCs of three -/- homozygous individuals. The blot was probed with antibodies generated against the carboxy terminus of Δ32 that specifically detect Δ32 protein. Over-exposure did not show any Δ32-related band in +/+ PBMCs. Samples (-/-) 1 , 2 and 3 on this blot correspond to #2, #3, and #6 respectively on Figure 12. Equivalent gel loading was verified by staining a similar gel with commassie blue stain.
CCR5 and Δ32 mRNA levels were analyzed in -/-, +/+ PBMCs using RT-PCR. Representative patterns of the amplified products are shown in Figure 16. The same RT-PCR analysis was performed using the same primers PHS 398. Figure 16 shows the expression analysis of Δ32 mRNA in -/- PBMCs Figure
16(A) and recombinant Ad5-infected cells Figure 16(B) by RT-PCR. Total cellular RNA was prepared from equivalent number of cells of each PBMC sample or Ad5- infected sample using QIAGEN- RNA isolation kit according to the manufacturers instructions and single step RT-PCR was done using Superscript one step RT-PCR kit (Gibco-BRL) by using CCR5-specific primers. Initially cDNA was prepared by incubation of the reaction mixture (total-50>ϊl) at 50°C for 30 minutes followed by one PCR cycle of 10 minutes at 94°C. This was subsequently followed by additional 34 cycles of PCR (94°C, 60 seconds; 65°C, 60 seconds;72°C, 90 seconds) and one cycle of 72°C for 10 minutes. The reaction products (10ml) were separated on 1% agarose in the presence of 1mg/ml ethidium bromide. Upstream and downstream oligonucleotide primers for amplification were: 5'-TGTGAA GCAAATCGCAGCCC-3' and 5'ATGGTG AAGATAAGCCTCACAGCC-3'. The primers were designed to amplify a 615bp Δ32 fragment and a 647bp CCR5 fragment. These experiments confirm expression of native mRNA in -/- individuals.
Samples 1 and 2 in Figure 12(A) correspond to samples 1 &2 in Figure 16. N represents a normal non-genotyped PBMC sample. Southern blot analysis was performed on the gel shown in Figure 16(A) and probed with a 32 P-labeled CCR5/Δ 32-specific DNA Figure 16(C).
These CCR5 and Δ32-related fragments could have been generated from smaller mRNA species. Previous studies on the analysis of CCR5 regulatory region indicated the presence of multiple CCR5 transcripts with 5'- end heterogeneity (Mummidi, et al.). It is interesting that sample#1 (-/-) contained low levels of the smaller fragment compared to sample #2 (Fig.16C). This result indicates that different patterns of Δ32 mRNA expression can exist in different Δ32 mutation carriers.
The analysis revealed abundant expression of Δ32 mRNA in -/- PBMCs. Expression of CCR5 mRNA in +/+ PBMCs was verified by detecting the correct size amplification product. Further, the ethidium bromide stained gels demonstrate that the Δ32 mRNA levels made by the vLA-2 are not higher than those found in vivo. This analysis confirms the abundant expression of Δ32 mRNA in -/- PBMCs and establishes that Δ32 protein levels attained by the Ad5 vector system are comparable to those made in PBMCs. This RT-PCR analysis was repeated with β- actin to control for the amount of RNA loaded in each lane and the results revealed that approximately equivalent amounts of RNA were used. The results demonstrate that Δ32 expression levels made by Ad5 vector system are comparable to those made in -/- PBMCs. To examine whether expressing Δ32 protein in cells reduces their susceptibility to HIV-1 infection by X4 viruses, MAGI-CCR5 cell line (obtained from the NIH AIDS Reagent Program) was used as target. MAGI-CCR5 cells express the human chemokine receptors CCR5 and CXCR4 and are therefore, susceptible to both macrophage-tropic and T-cell line adapted HIV-1 viruses (Kimpton, et al.). Cells were plated in 96-well plate and infected by vLA-1 (CCR5) or vLA-2 Δ32) viruses for 48 hours then infected with the X4 HIV-1 IIIB (obtained from the AIDS Reagent Program, NIH). As negative controls, MAGI-CCR5 cells were infected with a recombinant Ad5 encoding T7 RNA polymerase (provided by Frank Graham, McMaster University) at 27 pfu/cell or with a recombinant adenovirus encoding MVHA (Alkhatib, et al.) at 27 pfu/cell and assayed at the same time for PHS 398.
Figure 17 shows the effect of recombinant Δ32 protein on X4 infection. MAGI-CCR5 cells were infected with vLA-1 (encoding CCR5) or vLA-2 (encoding Δ 32 protein) at increasing viral concentrations (PFU/cell) then infected with the X4 IIIB. HIV-1 infection is quantitated by the amount of β-galactosidase produced as a result of HIV LTR activation that controls expression of β-galactosidase. The infection protocol is adapted from Kimpton and Emerman. Values of β-gal production in cells infected with Ad5pol3 (encoding T7 RNA polymerase) or Ad5/MVHA at 27 pfu/cell were 68 and 60 respectively. In other experiments, the R5 lab adapted isolate Ba-L was used and a similar inhibition curve was obtained.
The ability of recombinant Δ32 to provide protection against HIV-1 productive infection in vitro was examined. The experiment was performed by using (vLA-2 Δ 32-encoding adenovirus) or, as a negative control, vLA-1 (CCR5-encoding adenovirus) at a moi of 3 pfu/cell. Infected cells were incubated for two days to allow expression of recombinant proteins then infected with either an X4 or a R5 HIV-1. Under these experimental conditions, a pronounced reduction in HIV-1 productive infection was observed (>50% with IIIB Vs 40% with Ba-L; Figure 18). The data confirm the ability of Δ32 to provide protection against R5 as well as X4 infection of human PBMCs and suggests a possible interaction of Δ32 with CCR5 and CXCR4. In this experiment the Δ32 was delivered to PBMCs using Ad5/Δ32 at 3 pfu/cell and a 50% reduction in infectivity was observed. The efficiency adenovirus vector depends highly on the kind of cells used and a careful standardization of gene delivery into the PBMCs is necessary to perform in order to determine whether full resistance to infection can be obtained with higher doses of Δ32.
Figure 18 shows the Phytohemagglutinin-A+IL2-activated Ficoll-purified human PBMCs which were infected with either vLA-1 (Ad5/CCR5) or vLA-2 (Ad5/Δ 32) at 3pfu/cell for each virus for two days then infected with either Ba-L (R5) or IIIB (X4). Infections were performed in a 96-well plate. The virus was absorbed for three hours and cells were washed three times with PBS and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100mU of recombinant IL-2 and 10mg/ml PHA. Culture fluid (50 μl) was harvested after cell resuspension every three days and replaced with fresh medium. The amount of p24 antigen in the cell- containing supernatants was measured using an Elisa kit purchased from DuPont. The AZT control infection resulted in p24 values below 0.5 ng/ml.
Although the first line of protection in the Δ32 homozygote phenotype is the lack of expression of the required CCR5 coreceptor for virus infection, there is clear evidence of additional protective characteristics of the Δ32 heterozygous condition and the data has profoundly expanded on the roles and mechanisms of this genotype condition.
The experiments described herein highlight an important role of Δ32 in genetic resistance to HIV-1 infection. Recombinant Δ32 not only specifically downregulates expression of wild-type CCR5 confirming data in the literature, but have shown a specific and dramatic down-regulation effect on the other principal coreceptor CXCR4. CXCR4 reduction translates into specific protection from infection by CXCR4-using X4 viruses. Figure 19 summarizes this central concept.
Figure 19 shows the model for the Δ32 effect. In this model Δ32 protein interacts specifically with the major HIV coreceptors interfering with their proper transport to the cell surface. The impairment of coreceptor molecules reduces their chance to associate with CD4 leading to unfavorable stoichiometry for virus entry. The Δ32 effect is concentration-dependent. Higher Δ32 levels can correlate with less coreceptor and reduced virus entry. This concentration effect explains why +/- carriers show partial protective phenotype. Infected -/- cells either expresses low levels of Δ32 or expresses a defective Δ
32. The central finding of the data shows that recombinant Δ32 downregulates not only CCR5 but also CXCR4. In this model, Δ32 does not abolish CXCR4 but reduces its expression levels causing impairment of coreceptor utilization. There can be another unknown mechanisms for Δ32 activity that require the activity of another cellular protein. This mechanism is based on the data presented in Figure 8. The fact that CXCR4 is affected by Δ32 activity is relevant to disease progression and pathogenesis since CXCR4-using viruses emerge at the late symptomatic stage of the disease.
Understanding how Δ32 interacts with the major coreceptors leads to novel findings concerning coreceptor structure-function that can be utilized for drug design that mimic Δ32 activity.
The data provides evidence for the Δ32 effect on X4 fusion/infection. This experiment examined whether Δ32 protein colocalizes with the major coreceptors and provide evidence for their physical association using protein heterodimerization, co-immunoprecipitation and the yeast 2-hybrid system. These experiments set the stage for experiments focused at analyzing the structural determinants involved in Δ 32-coreceptor association. Many integral membrane proteins form noncovalently associated oligomeric complexes that are prerequisites for transport and normal function. Previous studies suggested that CCR5-truncated molecules similar to authentic Δ32 molecule (Figure 2) form heterodimers with CCR5 and was proposed as a molecular explanation for the delayed onset of AIDS in +/- individuals (Benkirane, et al.). This mechanism explains inhibition of CCR5- PHS 398.
The present study is the first to utilize the Δ32 open reading frame containing the frame shift 31 amino acid residues that are not expressed in CCR5.
Confocal microscopy was used to detect Δ32-coreceptor complexes using fluorescently labeled molecules. Cells expressing endogenous CCR5 and CXCR4 (i.e. MAGI-CCR5 cell line) was infected with Ad5/Δ32, examined for the Δ32 effect (downmodulation effect, as in Figure 4) and used to examine whether Δ32 colocalizes with CCR5 or CXCR4. Cells were either fixed or permeablized, and incubated with anti-Δ32 and FITC-labeled secondary antibody. Cell samples were then reacted with either anti-CCR5 or anti-CXCR4 monoclonal antibodies and a phycoerythrin-labeled secondary antibodies. Confocal laser scanning microscopy was performed to detect colocalization of the proteins.
Recombinant Ad5 encoding Δ32 or either of the major coreceptors was used to co-infect NIH-3T3 cells. At the optimum time, post-infection, total cell lysates were prepared and immunoprecipitated using the anti-CXCR4 antisera (Feng, et al.) or anti-CCR5 antibodies. Immnuoprecipitation with an antibody followed by western blotting with a different antibody provides a way to determine whether hetero- oligomers form. The immunoprecipitates were fractionated in 10%SDS-PAGE and transferred onto nitrocellulose membranes.
The blots were initially probed with anti-Δ32 antibodies to verify the presence of Δ32 protein, then stripped and reprobed with an antibody specific to either CXCR4 or CCR5. Since the Δ32 protein has a lower molecular weight (28-30KD) compared to CXCR4 or CCR5 (45-50KD), it was possible to distinguish the Δ32-CXCR4 or Δ 32-CCR5 heterodimers from the CXCR4 or CCR5 homodimers on the immunoblot. Monoclonal antibodies specific to Δ32 protein helped overcome some of the technical difficulties associated with the detection of Δ32-CXCR4 complexes. Co- immunoprecipitation was used as an alternative approaches to confirm the existence of Δ32-coreceptor complexes. Cells co-expressing Δ32 and CCR5 or CXCR4 (recombinant or endogenous) was used for immunopricipitation with anti-Δ32 antibodies. The immune complexes were fractionated and then immunoblotted. Co- precipitated CCR5 or CXCR4 was detected using specific antibodies to either protein.
Yeast two-hybrid systems provide a sensitive method for detecting transient protein interactions that are biochemically detectable. The system sensitivity allows quantitative analysis of mutant constructs to be used in the mapping studies. The system has been previously used to analyze CCR5 truncated molecules with wild- type CCR5 (Benkirane, et al.).
A MATCHMAKER two-hybrid system purchased from CLONTECH was used to analyze Δ32-co receptor interaction. The cDNA encoding Δ32 ORF was cloned into pGBKT7 (CLONTECH) while the coreceptor cDNA was cloned into pGADT7 (CLONTECH). Yeast AH109 (or Y187) was cotransformed with pGBKT7-Δ32 and pGADT7-coreceptor. The cotransformation mixture was plated on SD/-Leu/-Trp to select for colonies containing both hybrid plasmids. A colony-lift β-galactosidase assay was used as the system PHS 398.
X-gal staining of transformed colonies detects protein interaction. Quantitative analysis of the interaction was performed using 5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside as a substrate in liquid cultures. The amount of β-galactosidase made as a result of protein/protein interaction was quantitated using a colorimetric method. Parallel cotransformation controls using pGBKT7-Δ32 and either pGADT7- CXCR2, pGADT7-CD4, or pGADT7-CD44 was performed to control for the background of the system. The preliminary data indicated that Δ32 has no downmodulation effect on CD4, CXCR2 or CD44 (Figurel O) and therefore, was used as negative controls for coreceptor-Δ32 interactions.
Δ32 protein interacts with and specifically inactivates functional expression of other HIV coreceptors. Although CCR5 and CXCR4 are the major HIV coreceptors, a growing body of evidence suggests that other coreceptors have a role in HIV pathogenesis in certain tissues. For example, CCR3 plays a role in HIV pathogenesis of the brain (He, et al.). Possible contribution of other coreceptors to HIV pathogenesis of other tissues has been recently reported (Lee, et al., Sharron, et al., Faure, et al.). The result of this analysis explains why other coreceptors in -/- individuals are not utilized in the absence of CCR5. The effect of Δ32 on nonhuman CXCR4's were examined to analyze the specificity of Δ32 activity.
Cells coexpressing Δ32 and CCR5, CXCR4, CCR3, STRL33/BONZO, CX3CR1 , LCR1 (rat CXCR4) or AJ13 (sheep CXCR4) were examined in a fusion assay for their abilities to fuse with effector cells expressing the different HIV-1 Envs. CD4+ NIH-3T3 cells were coinfected with recombinant adenoviruses encoding Δ32 and the appropriate coreceptor (or a chemokine receptor) and Ad5pol3 (T7 RNA Polymerase). Effector Hela cells was coinfected with vCB-21 R (T7lacZ) and one of the HIV-1 Envs-expressing vaccinia vectors. The two cell populations was then mixed and screened for their ability to fuse. Data was collected using both the bulk assay for β-galactosidase reporter activity and scoring the syncytia formation ability of each Env. This assay was used to quantitate the relative effects of Δ32 on fusion activity mediated by the major and the minor coreceptors. In order to determine whether Δ32 action is specific for human HIV coreceptors, the coexpression experiments described above was performed using non-human CXCR4 homologs that share considerable amino acid homology with human CXCR4. The effect of Δ32 on coreceptor activity of CCR3 is particularly interesting because of the broad specificity of CCR3 for fusion with X4 as well as R5 HIV-1 isolates. The preliminary, studies Jiave. relied largely on antibodies generated against wild type CCR5 (PDL) to detect both CCR5 and Δ32 proteins. Although these antibodies are reactive with recombinant Δ32 in some assays (i.e. sandwich ELISA), it is critical to be able to detect the Δ32 in cells co-expressing Δ32 and wt CCR5 and/or CXCR4. Δ32 was not detected in immunoblots using commercially available CCR5 Mabs raised against the CCR5 N-terminus (Figure 14A). However, polyclonal antibodies directed against CCR5 N-terminus and Δ32 C-terminus were successfully used to detect Δ32 protein (Figure 14B and C).
The recombinant adenovirus expressing Δ32 protein was used as an antigen to immunize the mice. This provides a method to deliver Δ32 in its native conformation. These experiments take place in the animal colony of Indiana University medical center. The generated MAbs was used as reagents to detect Δ32, and to map its functional domain(s) using a panel of point mutants. Since Mabs were generated using the vLA-2 that delivers native Δ32, generated antibodies had different specificities that helped map critical epitopes in Δ32. This was accomplished by examining the ability of Δ32 variants (chimeras and point mutants) to dbwnmodulate coreceptors and be recognized by a particular Mab.
Monoclonal antibodies took some time to generate. Once the Δ32 point mutants and chimeras (Section D2c), were generated, the use of Mabs contributes significantly to the structure-function studies.
Δ32 protein impairs the formation of CD4-Coreceptor complexes by reducing coreceptor availability. Coreceptors compete for association with CD4 (Lee, et al.) Δ 32 protein reduces this interaction by associating with the coreceptor.
CD4-coreceptor complexes exist without the presence gp120 and by using confocal microscopy the colocalization of such complexes has been demonstrated
(Xiao, et al.). The same study demonstrated that CD4-CCR5 complexes are stronger than CD4-CXCR4 complexes. It is not known however, whether CD4- coreceptor association occurs cotranslationaly or at the cell surface.
Recent studies by Lee, et al. demonstrated that X4 or R5 fusion is reduced upon introduction of CCR5 or CXCR4 respectively by vaccinia virus vectors.
Applicants also demonstrated that introduction of high levels of CD4 on human T cells can reverse the ability of CCR5 to reduce X4 fusion and postulated that CXCR4 and CCR5 compete for association with CD4 (Lee, et al.). In the system, introduction of CCR5 into cells expressing endogenous CD4 did not result in significant reduction of X4 fusion (Figures 11 and 17). These discordant results could be due to the different viral vectors used to express coreceptors, different cell types, or/and the method used to collect the data (fusion was quantitated by intracellular activation of β-gal while Lee, et al. by counting the number of syncytia). The data demonstrated that Δ32 causes a dramatic reduction of surface CXCR4 and CCR5 but does not abolish their expression completely (For example, see Figure 4). The question is whether the presence of high levels of CD4 can reverse the Δ32 effect on X4 or R5 fusion. A similar approach reported by Lee, et al. was followed to examine the effect of CD4 expression on Δ32 activity. CD4 levels are not affected by Δ32 in several cell types.
Hela cells were used in this analysis because it expresses endogenous CXCR4. A CD4-encoding vaccinia virus using different doses of the virus delivers increasing levels of CD4. These expression levels were established by infecting Hela cells with 1 pfu/cell (low), 5 pfu/cell (medium) or 20pfu/cell (High). Cells were verified for CD4 expression by quantitative FACS analysis. The Δ32 protein was introduced into these cells by Ad5/Δ32 at an inhibitory dose (10-20pfu/cell) and challenged with X4 or R5 Env-expressing cells. The effect of high CD4 levels delivered by the vaccinia vector was assayed by quantitating X4 and R5 fusion compared to control virus-infected cells.
If high levels of CD4 did not reverse Δ32 effect, then it suggests that Δ32 can have higher affinity for CXCR4 than CD4. However, if high CD4 reversed the Δ32 effect it suggests the opposite and results in competition between with Δ32 and CD4 over newly made CXCR4 molecules during cellular trafficking.
If CD4 associates with coreceptor cotranslationally, then it is possible to colocalize the CD4-co receptor Δ32 by confocal microscopy. A similar approach was taken to study the effect of CCR5 and CXCR4 concentrations on Δ32 effect in cells expressing limited amounts of CD4. This analysis determines whether the ratio of pre-existing CD4-CCR5 to CD4-CXCR4 complexes influences Δ32 effect. Cell lines such as MAGI-CCR5 were suitable for this kind of analysis since it contains all molecules involved in the process. The levels of CD4 expressed by vaccinia virus vectors are very high and may not be relevant to the in vivo situation. Additionally, high multiplicity infection can change membrane structures leading to high background levels. As another approach cell lines expressing different levels of CD4 were used and assayed for its effect on intracellular Δ32.
The preliminary data demonstrated that recombinant Δ32 caused downmodulation of not only CCR5 but also CXCR4 showing that it interacts with the major coreceptors using similar structural determinants.
Although CCR2 shares more than 75% homology with CCR5 Alkhatib, et al., it was not downmodulated by Δ32 suggesting a possible specific interaction that involves at least, part of the non-homologous region.
However, downmodulation of CXCR4, a CXC chemokine receptor that only shares 30% homology with CCR5, suggests a common structural determinant.
Moreover, Δ32 expression has no significant effect on surface levels of CD4 confirming that the observed reduction in X4 fusion/infection is not due to an effect on CD4.
CCR5/CCR2 hybrid constructs and CCR5 point mutants (Alkhatib, et al.) were also utilized to analyze the region of CCR5 that is likely to interact with Δ32. Similarly, CXCR4 point mutants were utilized to determine a potential region of CXCR4 that interacts with Δ32.
The unique carboxy terminal 31 amino acids in the Δ32 protein create new functional domain(s) that can be specific for interaction with coreceptors.
A direct way to determine whether the carboxy terminal region of Δ32 is critical for its activity is to construct chimeric molecules where this unique Δ32- specific COOH tail or portion of it is grafted onto CCR5 that lacks its carboxy tail. CCR5 lacking the carboxy terminal cytoplasmic tail is expressed at the cell surface and is functional as a coreceptor (Alkhatib, et al.). Co-expression of tail-less CCR5 and wild type CCR5 or CXCR4 had no effect on R5 or X4 fusion, which is in agreement with published data using a similar construct (Benkirane, et al.). If the biological activity of Δ32 is contained within the last 31 amino acids at its carboxy terminus, then adding this region to a tail-less CCR5 is expected to result in the loss of its ability to reach the cell surface. This analysis is extended to include other truncation of this Δ32 fragment in order to map its interactive domain. Once the Δ32 inactivating domain is identified by chimeric constructs, further site-directed mutagenesis strategies are employed to obtain a more detailed map of the critical amino acid residues required for Δ32-induced activity. Mutant constructs are then used in the reporter gene activation fusion assay to study their effect on cell surface transport and coreceptor activity.
Mutagenesis of the Δ32-specific 31 amino acids is not sufficient to reveal the critical domain of this protein, however, since these amino acids are not found in CCR5. Depending on the data obtained using the first group of mutants that target the Δ32-specific segment, mutants can be designed to include the shared homologous region with CCR5.
Since CXCR4 and CCR5 share only 30% homology (including conserved amino acid changes), it is important to examine whether a common region of the principal coreceptors CXCR4 and CCR5 is responsible for interacting with Δ32. Knowledge of this common region provides insight into the molecular basis of coreceptor activity and the design of new drugs that can have a broader antiviral effect.
A direct method to map important regions of coreceptor involved in interaction with Δ32 can be to construct chimeric molecules between CXCR4 or CCR5 with homologs that do not function as coreceptors or show a different coreceptor function in terms of Env specificity (i.e. CCR2 works only with 89.6 but not with R5 Envs).
The preliminary results indicated that Δ32 has no downmodulation effect on CXCR2 or CCR2 expression (Figure 10). These two receptors are the most homologous to CXCR4 and CCR5 that show a dramatic Δ32- induced downmodulation effect. This criteria is used to map the region of the CCR5 or CXCR4 that interacts with Δ32.
This is a complementary experimental strategy to that outlined previously. Here, use of hybrid CCR2/CCR5 and CXCR2/CXCR4 constructs are used to map potential regions of the major coreceptors that interact with Δ32. There were previously described coreceptor activities of chimeric constructs of CCR2/CCR5 (Alkhatib, et al.). A panel of CXCR2/CXCR4 chimeric molecules was obtained from the laboratory of Dr. Stephen Pieper, University of Kentucky. The mutants include a number of exchanged receptor segments (i.e. 2444, 2244, 2224, 4222, 4422, and 4442). The hybrid molecules are transfected into 293 cells followed by infection with Ad5/Δ32 and FACS analysis to determine the hybrid molecules that lost the ability to associate with Δ32. The loss of function is characterized by the ability of the hybrid molecule to escape the effect of Δ32 indicative by its efficient cell surface expression. Obtaining functional mutants is also useful to verify and confirm the interactive region.
Using this approach, one is able to identify the coreceptor region by identifying gain of function and loss of function chimeras. Once that coreceptor region is mapped, a panel of CXCR4 point mutants (provided by Chris Broder (Chabot, et al.)) as well as CCR5 point mutants (Alkhatib, et al.) are used to determine the amino acid residues involved in interaction with Δ32. Most CXCR4 point mutants are expressed at the cell surface and therefore, are assayed for the Δ 32 effect by FACS analysis.
Surface modulation is not be sufficient to classify mutants and therefore, a more sensitive and quantitative assay such as the yeast two-hybrid system is used as an alternative method to detect changes in the interaction of coreceptor mutants with Δ32.
Antibodies to Δ32 are now available and are being used to specifically detect Δ32 protein in Western blots and immunoprecipitation. One first determines whether Δ32 physically associates with the major coreceptors. The biological analyses of chimeric mutants as well as the heterodimer formation experiments can initially use the polyclonal antibodies for detecting the mutant protein. Once the Mabs are made they are used with Δ32 chimeras and point mutants to map the critical determinants of this protein.
The protective effect of the Δ32 mutation failed in 8 homozygous individuals (Michael, et al.). It remains unknown why some -/- individuals become infected with HIV-1. These individuals may not express Δ32 and therefore, have lost the protective effect of this naturally occurring mutation. Alternatively, the infected -/- expresses a defective Δ32 molecule. Comparative analysis of Δ32 mRNA and protein in protected versus unprotected -/- PBMCs provides important information. Moreover, a comparative analysis of unstimulated PBMCs isolated from infected and uninfected -/- individuals is performed to quantitate Δ32 and coreceptor expression. Figure 16 shows that it is possible to detect the Δ32 protein in unstimulated -/- PBMCs. It is also possible to detect CXCR4 mRNA and protein expression in unstimulated PBMCs (Figure 9).
Recent findings demonstrated that Δ32 mutation was always associated with a CCR5 G/A promoter polymorphism in +/- heterozygotes (Ginocchio, et al.). These promoter mutations influence CCR5/Δ32 mRNA production in these individuals leading to different levels of Δ32 mRNA and protein.
Some support is provided from studies by Ometto, et al. who proposed that CCR5/Δ32 heterozygosity confers a different degree of protection against HIV-1 in PBLs and MDMs, depending on the ratio of wild-type and mutant Δ32 mRNA in the two cell types. The investigators analyzed expression levels of wild type and mutant Δ32 mRNA species by competitive RT-PCR and demonstrated that depending on the cell type, Δ32 heterozygosity confers a different degree of protection against HIV-1 infection.
To avoid the discrepancy in the literature in terms of CXCR4 expression levels in activated PBMCs, unstimulated cells are used to quantitate Δ32 and CXCR4 levels. The final goal of this analysis is to provide an in vivo evidence for expression levels of CXCR4 in resting -/- and +1- PBMCs and whether they correlate with endogenous levels of Δ32 protein. Quantitative analysis of Δ32 mRNA expressed in -/- and +/- PBMCs is performed using a competitive RT-PCR and protein analysis methods (Figure 14&15) in order to determine whether levels of Δ32 in different PBMCs correlate with lower expression levels of CXCR4.
Antibodies raised against the COOH-terminus of Δ32 to analyze its expression in PBMCs of -/- and +/- individuals were used. A number of -/-, +/-, and +/+ PBMCs (Zimmerman, et al.) are used. Cell lysates of unstimulated PBMCs, or PHA-stimulated PBMCs from homozygotes and heterozygotes are prepared and analyzed for expression of endogenous Δ32 protein by immunoblotting and immunoprecipitations as presented in the preliminary data section. Quantitative protein analysis are performed to determine whether there are differences in Δ32 protein levels in -/- versus +/- individuals that can explain the partial, incomplete protective phenotype in heterozygotes. Similar analysis are performed on the infected -/- PBMC sample to determine whether Δ32 protein is expressed in these cells.
PBMCs from one infected Δ32 homozygote is not representative of all 8 homozygotes identified so far (Michael, et al.). Unfortunately, some of these individuals are dead. and it is possible to examine their samples.
PBMCs from -/- individuals provide an in vivo system where the effect of Δ32 on endogenous coreceptors can be assayed in their native environment. Δ32 protein is expressed and can be detected in -/-PBMCs (Figure 15). It is important to examine Δ32-CXCR4 interaction in -/- PBMCs to provide an in vivo evidence for this molecular association. As mentioned earlier, the association of Δ32 and CXCR4 is relevant to pathogenesis.
Pulse chase experiments are performed to compare coreceptor protein synthesis in -/- and +/+ PBMCs to directly examine biosynthesis and stability of coreceptor proteins made in these two cell populations. Unstimulated as well as PHA+IL2 stimulated -/- and +/+ PBMCs are used for labeling with 35 S-methionine.
Activated cells express higher levels of endogenous proteins and therefore can be useful in tracing coreceptor proteins in pulse-chase experiments. However, the protein analysis shown in Figure 15 indicated the detection of Δ32 protein in unstimulated cells. To detect Δ32-CXCR4 complexes, cell lysates are prepared and used in co-immunoprecipitation assays. The effect on stability of CXCR4 in -/- versus +/+ PBMCs is examined in pulse chase experiments followed by immunoprecipitation of CXCR4 with monospecific antibodies (Feng, et al.). These experiments reveal differences in CXCR4 biosynthesis and processing in -/- and +/+ cells and provide an in vivo evidence for the existence of Δ32-coreceptor complexes. Figure 8 shows that Δ32 caused downmodulation of CXCR4 in 293 cells and MAGI-CCR5 cell line but not in HL60 cells indicating that Δ32 activity can be cell- type specific (Fig.8A).
The experiment is focused on developing a cellular model system to examine other factors that can contribute to resistance to HIV-1. Identification of these factors opens new avenues in this field and has important implications for understanding the molecular basis of resistance to HIV-1.
The human myeloid HL-60 cell line can be differentiated into macrophages upon treatment with RA. Upon RA treatment, these cells differentiate to acquire a macrophage phenotype and become fusogenic with R5 tropic HIV-1 (Alkhatib, et al.). Since the Δ32 effect is observed in HL60 differentiated cells, then it suggests expression of another cellular protein that can be critical for the Δ32 effect. First, one must determine whether Δ32 can downmodulate CXCR4 in RA-treated HL60 cells using untreated cells as a negative control. Subtraction cDNA library is made using undifferentiated and RA-differentiated cells as a source of RNA. If the induced molecule(s) is required for Δ32 then it can interact with it. A direct method to examine and isolate the interacting protein is using the yeast two-hybrid screening system. Once the cDNA is/are isolated, it is transfected into untreated HL60 cells to examine if it can transfer the Δ32 effect.
Subsequently, the cDNA clone is sequenced and its identity and homology with other proteins are analyzed. The induced molecules in RA-treated HL60 cells cannot interact with Δ32 but can interact with CXCR4. Another two-hybrid screening is performed using CXCR4 as the bait protein. If these screening methods failed to identify the induced molecule, a functional cDNA screening method is used to isolate the cDNA clone. This method is based on the transfer of Δ32 function to untreated HL60 cells. Briefly, the cDNA library under T7 promoter is transfected into HL60 cells, followed by infection with Ad5pol3 (T7 RNA pol) and Ad5/Δ32. Reduction in X4 fusion indicates transfer of function. The cDNA library is then subfractionated and fractions are used to repeat the screening until a single clone is identified.
Cell lines transformed with Δ32 provide a cellular model for the analysis of Δ 32 activity and its contribution to resistance to HIV-1. Creation of this cell line is critical to analyze Δ32-CXCR4 complex formation in cells synthesizing endogenous levels of Δ32 and CXCR4 proteins. This is a Δ32-CXCR4 interaction without the presence of endogenous CCR5. This allows studying the effect of introduction of exogenous CCR5 in order to determine whether the major coreceptors compete for complexing with Δ32. The analysis of Δ32 has relied on Ad5 vectors to express recombinant Δ32 in cell lines as well as primary cells. In all experiments the Δ32 gene was introduced into cells expressing endogenous CXCR4 and/CCR5.
The preliminary data shown in Figures 17 and 18 indicated that cell lines as well as PBMCs expressing recombinant Δ32 showed reduced susceptibility to HIV-1 infection. It is of basic interest to examine Δ32 effect in a CD4+ T-cell line that expresses endogenous levels of Δ32 protein. Jurkat cells are CD4+CXCR4+ and endogenous expression of Δ32 directly address its effect on endogenous CXCR4 expression and susceptibility to infection. This allows detailed analysis of the intracellular distribution and localization of Δ32 protein and its possible interaction with CXCR4. The choice of Jurkat cell line is based on the fact that it is CD4+CXCR4+ and it shows Δ32 effect when infected with Ad5/Δ32.
Standard procedures are used as previously described to create a Jurkat- CCR5 cell line (Alkhatib, et al.). Positive clones are identified by PCR on cellular genomic DNA. Clones are selected on the basis of the Δ32 made using RT-PCR as a method of quantification. Clones with low, medium, and high levels of Δ32 mRNA expression are selected and expanded. Antibodies to Δ32 can finally be used to identify the clones expressing low, medium, or high levels of the Δ32 protein. Once clones of Δ32 cell lines are purified they can be tested in infectivity assays to determine the percent reduction in HIV-1 fusion/infection assays. The selected clones expressing different levels of Δ32 are extremely useful in determining whether a dose-effect correlation can exist in vivo.
Cytotoxicity can be a problem, however, this was not observed with CCR5 or/and CXCR4 cell lines. A number of cell types can be tested at the same time and select the one that shows better progress in terms of Δ32 expression can be selected. Example 2: The Yeast Two-hybrid system was used to provide evidence for CXCR4 interaction with the Delta-32 protein.
Matchmaker two-hybrid system 3 (GAL4-based) was used to study the interaction of CCR5/CXCR4 protein with Delta-32. The system provides a transcriptional assay for detecting protein interactions in vivo in yeast. Delta-32 gene was expressed as a fusion to the GAL4 DNA-binding domain (DNA-BD), while CCR5 or CXCR4 was expressed as a fusion to the GAL4 activation domain (AD). In the present study DNA-BD fusion vector, pGBKT7 and AD fusion vector pGADT7, were used for high level expression. Delta-32 (cloned in pGBKT7) and CCR5/CXCR4 (cloned in pGADT7) inserts were expressed as GAL4 fusions with c- Myc and hemagglutinin (HA) epitope tags, respectively. The transcription and translation of epitope-tagged fusion proteins in vitro was driven by T-7 promoter, which is at the downstream of the GAL4 coding sequence.
Cotransformation was carried out using both the bait -Delta-32 and AD fusion vector-CCR5/CXCR4 in yeast strain AH 109, which is gal4" and gal80" prevents interface of native regulatory proteins with the regulatory elements in the two-hybrid system. pGBT7-53 and pGADT7-T encode fusions between the GAL4 DNA-BD and murine p53 and SV40 large T-antigen, respectively. p53 and large T-antigen interact in a yeast two-hybrid assay and was used as positive control. PGBKT-7-Lam encodes a fusion of the DNA-BD with human lamin C and provides a control for a fortuitous interaction between an unrelated protein and the pGADT7-T and was used as a negative control. Results and Observations:
Figure imgf000047_0001
Exp.#1 is a negative control. Exp.#2 is a positive control for interaction of two well-known proteins. Exp. #3, 4, and 5 are all controls to measure the background of the system. Exp.#6 and 7 represent confirmed results of Δ -32 protein interaction with not only CCR5 but also CXCR4. Interaction of Δ-32 with CXCR4 is a novel finding that has never been described in the field of AIDS. This finding is significant because CXCR4 is linked to disease progression and represent an important coreceptor that the majority of HIV-1 isolates utilize during the symptomatic stage of AIDS. Example 3:
Human immunodeficiency virus type 1 (HIV-1 ) employs CD4 and a coreceptor, principally the CCR5 and/or CXCR4 chemokine receptors, for entry into host cells. The central role of CCR5 in HIV-1 transmission and pathogenesis has been highlighted by the epidemiological and genetic identification of powerful disease-modifying effects of the naturally occurring CCR5Δ-32 (D32) allele, a 32- base pair deletion encoding a truncated and non-cell surface expressed version of CCR5. Relative to the general population, D32/D32 homozygotes are rarely found among HIV+ individuals but are significantly more common among repeatedly exposed/uninfected (EU) individuals. Moreover, HIV+ CCR5/D32 heterozygotes progress more slowly to AIDS than individuals lacking this allele. Previous studies have shown that D32 binds to CCR5, retarding its transport to the cell surface. However, this mechanism does not explain why CXCR4 does not compensate for CCR5 deficiency in vivo. Methods:
By using a recombinant adenovirus encoding the delta-32 (D32) open reading frame (ORF), it was demonstrated that the D32 protein is expressed intracellularly towards the inner surface of the cell membrane. It was also demonstrated that D32- protein expression can specifically reduce expression of endogenous CCR5 and CXCR4 resulting in the inhibition of HIV-1 entry. This effect was not observed in cells expressing the full-length recombinant CCR5 or other non-relevant proteins. Deletion of the last 31 amino acid residues of D32 ORF abolished its CXCR4 down- modulation effect implicating a specific D32 domain critical for CXCR4 interaction. Confocal microscopy and yeast 2-hybrid techniques were used to confirm D32 interaction with CXCR4 and CCR5. Finally, expression of D32 protein (by a recombinant adenovirus) in human peripheral blood mononuclear cells (PBMCs) conferred broad resistance against infection by diverse HIV-1 isolates including prototypic X4 strains. Results: Using D32-specific antibodies, it was demonstrated that D32 protein is expressed in PBMCs isolated from EU individuals homozygous for the D32 allele. The protein encoded by D32 ORF down-regulates the major co-receptors in vivo resulting in a broad protection against disease transmission and progression. Conclusions:
This is the first study to analyze the native D32 protein at the molecular level and can have important implications for the design of drugs that can be effective against viral transmission and disease progression. Example 4:
Human immunodeficiency virus type 1 (HIV-1) employs CD4 and a coreceptor, principally the CCR5 and/or CXCR4 chemokine receptors, for entry into host cells. The central role of CCR5 in HIV-1 transmission and pathogenesis has been high-lighted by the epidemiological and genetic identification of powerful disease modifying effects of the naturally occurring CCR5Δ32 allele, a 32 base pair deletion encoding a truncated and non-cell surface expressed version of the coreceptor. Using a recombinant adenovirus encoding the Delta-32 (Δ32) open reading frame (ORF) and Δ32-specific antibodies we have found that the Δ32 protein is expressed inside the cytoplasm towards the inside surface of the cell membrane. We have found that Δ32 protein expression can specifically reduce the expression of endogenous CCR5 or CXCR4 resulting in the inhibition of virus entry and infection by R5 and X4 HIV-1 isolates. This effect was not observed in cells expressing recombinant CCR5 or other non-relevant proteins such as measles virus hemaglutinin (MVHA) or structural adenovirus proteins. We hypothesize that the Δ 32 protein down-regulates the major coreceptors resulting in an unfavorable stoichiometry of the molecules involved in viral entry. Using Δ32-specific antibodies, we have shown that Δ32 is expressed in Δ32/Δ32 PBMCs. We propose to determine the mechanism(s) that underlies this more broad protective effect of the expressed Δ32 protein. Specifically, the aims of the present proposal are to: 1) Analyze the mechanism of Δ32-impairment of coreceptor function, and 2) map the structural determinants responsible for Δ32 activity. Background and Rationale:
Infection by the human immunodeficiency virus type 1 (HIV-1) requires CD4 and a coreceptor. The chemokine receptors CCR5 and CXCR4 are the major coreceptors used by most R5 and X4 HIV-1 isolates. The importance of chemokine receptors in HIV-1 transmission is highlighted by the finding that individuals homozygous for a 32-base pair deletion in CCR5 (Δ32/Δ32) are resistant to HIV-1 infection. The defective coreceptor gene encodes a prematurely terminated protein (Fig.lA) that is not detected at the cell surface and therefore is not functional as a fusion coreceptor. We will refer to Δ32 homozygous as -/-, Δ32 heterozygous as +/- and to those with wild type CCR5 as +/+. Genotypic analysis of this mutation and its distribution revealed that Δ32 has a high allele frequency among Caucasians but was absent in African or Asian populations. The mutant allele is not associated with any obvious phenotype in uninfected homozygous individuals. Heterozygotes (+/-) are not protected against infection, but once they become infected, have a slower progression to AIDS, indicating that partial resistance can occur in the presence of a single copy of the mutant CCR5 gene. The frameshift Δ32 mutation introduces 31 new amino acid residues at the carboxy terminus of Δ32 that are not present in CCR5 (Fig. 1A&C).
The first molecular analysis of Δ32 activity utilized Δ32-like molecules lacking the C-terminal 31 amino acids of native protein to show that they interact with wt CCR5 forming heterodimers that are retained in the endoplasmic reticulum resulting in reduced cell surface expression. These findings suggested that CCR5/Δ32 heterodimerization is a molecular mechanism for slower progression to AIDS in individuals with a heterozygote genotype. Our preliminary data are in full agreement with those published by Benkirane et al in that Δ32 interacts with CCR5 retarding its cellular transport. Additionally, we report a novel finding on the ability of Δ32 protein to specifically downmodulate cell surface CXCR4 resulting in a dramatic reduction in its coreceptor activity. This proposal is focused on the analysis of the mechanism of Δ32-inpairment of coreceptor function. Mapping of the common structural determinants involved in the interaction of Δ32 with the major coreceptors will help design novel coreceptor-based inhibitors that may have broad antiviral effect. Experimental Studies: Detection of CCR5Δ32 protein in Ad5/Δ32- infected 293 cells:
The CCR5Δ32 protein was expressed and analyzed in 293 cells infected with a recombinant adenovirus Ad5/Δ32 (Figure 1 B) (which also express green fluorescent protein) using a 31 -amino acids custom peptide antibody generated against the carboxy terminus of Δ32-ORF (Figure 1C). The Δ32 protein was found to be abundantly expressed in 293 cells infected with Ad5/Δ32 and showed a protein band at around 28-30 kDa (Figure 2A &B)). The Δ32 protein was also detected using anti-CCR5 antiserum directed against the common N-terminus of CCR5 and CCR5Δ32. In permeabilized cells, immunostaining of Δ32 protein indicated that it is expressed intracellularly towards the inner side of the plasma membrane (Figure 2C). Specific Δ32-induced downmodulation of CCR5 and CXCR4 in PBMCs:
The effect of recombinant Δ32 was examined in PBMCs freshly isolated from healthy donors. PBMCs were stimulated with PHA+IL-2 for 3 days then used for FACS staining and cell fusion. The PBMCs were infected with Ad5/Δ32 and examined for Δ32 effect on cell surface levels of CCR5, CXCR4, CD4, CD25, CD44, and CXCR2 by FACS analysis. We had three different controls in this experiment that included PBMCs infected with Ad5/CCR5. AdδMVHA, or wild type Ad5 vector. Specific downmodulation of CCRδ and CXCR4 was readily observed in PBMCs expressing recombinant Δ32 protein but not in PBMCs expressing wild type CCRδ or Adδ-encoded proteins (Figure 3A-F). Our envelope (Env)/target assay involves the analysis of fusion between two distinct cell populations, one expressing < CD4 (endogenous or encoded by a recombinant virus) and the other expressing the HIV-1 envelope glycoprotein encoded by a recombinant vaccinia virus. We showed that expression of recombinant Δ32 protein in PBMCs resulted in the specific inhibition of Rδ and X4 HIV-1 Env-mediated fusion (Figure 3G&H) in a dose dependent manner. The degree of fusion inhibition was proportional to the amount of expressed Δ32 protein made in the infected PBMCs (Figure 3, protein blot). The gradual increase in the inhibitory effect was proportional to the increasing intensity of Δ32 protein band. In other experiments, we found that the levels of Δ32 protein obtained by our ADδ/Δ32 could not be obtained by simple DNA transfection of 293 cells (data not shown) which may explain the inability of Venketesan, et al. (Bleul, et al.) to observe the Δ32 effect.
To confirm the Δ32 specificity we showed that expression of recombinant Δ32 protein in CD4 target cells (MAGI-CCRδ) resulted in the specific inhibition of X4 HIV- 1 Env-mediated fusion but had no effect on HTLV-1 Env-mediated cell fusion (Figure 4). In another experiment, we confirmed previous data in the literature that Δ32 inhibits Rδ fusion/infection and demonstrated that truncated Δ32 constructs lacking the novel 31 C-terminal amino acids reduced Rδ fusion but had no significant effect on X4 fusion suggesting a critical role of the Δ32 C-terminal region in CXCR4 interaction (Figure δ).
Analysis of native Δ32 RNA and protein in +/+ and -/- PBMCs:
Delta-32 RNA and protein expression in (-/-) PBMCs was also examined. RNA expression of Δ32 was verified by RT-PCR analysis (Figure 6A). The immunoblot analysis revealed a protein band (Figure 6B&C) that corresponds to the same molecular weight band seen with recombinant protein analysis (Figure 2). These protein bands were not obtained with the preimmune serum. The identity of the band that appears above the 34 Kd marker band is not known at present, however, since it is expressed in normal CCRδ individuals, it could represent a cross- reactive cellular protein. Our analysis also revealed that the Δ32 protein band detected in the protected (-/-) individuals was absent in an infected (-/-) individual (Figure 6C) suggesting a critical role for the Δ32 ORF in resistance to HIV.
Recombinant Δ32 conferred a broad protective effect against X4 and Rδ HIV- 1 infection. We examined the effect of expressing the Δ32 protein in an HIV- negative healthy PBMC sample on HIV-1 infection. PHA+IL-2 stimulated PBMCs were infected with either Adδ/Δ32 or Adδ/CCRδ before they were challenged with IIIB (X4) or Ba-L (Rδ) HIV-1. The results demonstrated a dramatic reduction in p24 production in cells expressing Δ32 but not CCRδ (Figure 7) providing the first evidence for the transfer of the protective nature of this protein at the molecular level. Analysis of Δ32-coreceptor interaction:
Colocalization and yeast two hybrid studies were performed to show that Δ32 forms complexes with not only CCRδ but also CXCR4 (Figure 8&9). Matchmaker Yeast two-hybrid system 3 (GAL4-based) was also used to study the potential interaction of CCRδ/CXCR4 protein with Δ32. Delta-32 (cloned in pGBKT7) and CCRδ/CXCR4 (cloned in pGADT7) inserts were expressed as GAL4 fusions with c- Myc and hemaglutinin (HA) epitope tags, respectively. The results strongly suggested that Δ32 interacts with the major coreceptors (Figure 9) as summarized in table 1. The analysis also confirmed the loss of Δ32 interaction with CXCR4 upon deleting the C-terminal 31 residues. Summary of studies: Although the first line of protection in the Δ32 homozygote phenotype is the lack of expression of the required CCRδ coreceptor for virus infection, there is clear evidence of additional protective characteristics of the Δ32 heterozygous condition and our preliminary data has profoundly expanded on the possible roles and mechanisms of this genotype condition. The experiments described in this proposal highlight an important role of Δ32 in genetic resistance to HIV-1 infection. We have demonstrated that recombinant Δ32 not only specifically downregulates expression of wild-type CCRδ, but also show a specific down-regulation effect on the other principal coreceptor CXCR4 and show evidence that this CXCR4 reduction translates into specific protection from infection by CXCR4-using X4 isolates. Experimental design and procedures:
This section is divided into two parts, one for each specific aim. Recombinant vectors encoding Δ32 protein used to show the specific effect on surface expression and X4 and Rδ fusion/infection and control Adδ/CCRδ have already been constructed Polyclonal antibodies that specifically detect native and recombinant Δ 32 protein have been used to verify expression of this novel protein in PBMCs isolated from (-/-) individuals. The experiments examine the physical association of the major coreceptors with Δ32 protein, generate monoclonal antibodies to Δ32 protein and determine the subcellular localization of Δ32 protein/coreceptor complexes. Analyze how Δ32 impairs HIV coreceptor function and analyze physical association of Δ32 /coreceptor:
First, cell surface analysis of cells expressing either recombinant or endogenous Δ32 protein showed reduced levels of CXCR4 and CCRδ. Second, yeast 2 hybrid assay also revealed that Δ32 specifically interacts with CCRδ or CXCR4. Third, confocal microscopy studies demonstrated that in addition to CCRδ/ Δ32 colocalization, CXCR4 and Δ32 colocalize intracellularly.
Δ32 protein interacts with CXCR4 and CCRδ resulting in an unfavorable stoichiometry of the molecules involved in viral entry and providing a protective phenotype against infection by X4 and Rδ HIV-1 isolates. Experimental Strategy:
This aim is devoted to examine the Δ32/coreceptor physical association using protein heterodimerization, immunoblotting and the yeast 2-hybrid system. Total cell lysates expressing Δ32 and one of the major coreceptors will be prepared and I immunoprecipitated using anti-CXCR4 antiserum or anti-CCRδ antibodies. The blots will be initially probed with anti-Δ32 antibodies to verify its presence then stripped and re-probed with an antibody specific to either CXCR4 or CCRδ. Since the Δ32 protein has a lower molecular weight (28-30kDa) compared to CXCR4/CCRδ (45- 50KD), it will be possible to distinguish the Δ32-CXCR4 or Δ32-CCRδ heterodimers from the CXCR4 or CCRδ homodimers on the immunoblot. Anti-c-myc or anti-HA antibodies will also be used for immunoprecipitating Δ32 with CXCR4 or CCR5 in yeast two hybrid system as Δ32 and the coreceptors are expressed as fusion proteins of c-myc and HA. At present, we are using polyclonal antibodies to specifically detect recombinant and native Δ32 protein. Monoclonal antibodies to Δ32 protein may help overcome some of the technical difficulties associated with the detection of Δ32- CXCR4 complexes. Co-immunoprecipitation will be used as an alternative approaches to confirm the existence of Δ32-coreceptor complexes. Cells co- expressing Δ32 and CCRδ or CXCR4 (recombinant or endogenous) will be used for immunoprecipitation with anti-Δ32 antibodies. The immune complexes will be fractionated and then immunoblotted. Co-precipitated CCRδ or CXCR4 will be detected using specific antibodies to either protein. Generation of monoclonal antibodies (Mabs) against Δ32 protein: The preliminary studies have relied largely on antibodies generated against wild type CCRδ to detect both CCRδ and Δ32 proteins. Although the polyclonal antibodies we developed against the Δ32 are reactive with recombinant Δ32 protein, they also cross-react with another cellular protein(s) (Figure 3). We plan to generate Mabs to the 31 amino acid peptide that we used to generate the polyclonal antibodies. The Mabs will be useful in the analysis of Δ32 protein biosynthesis and processing. Experimental approach:
The recombinant adenovirus Adδ/Δ32 is used as an antigen to immunize the mice. These experiments take place in the animal colony of Indiana University medical center. This will deliver the Δ32 protein in its native conformation providing an opportunity to generate Mabs to native Δ32 epitopes. Examining the ability of Δ 32 variants (chimeras and point mutants) to downmodulate coreceptors and be recognized by a particular Mab will help generate a functional map of Δ32. By generating a series of Mabs and continuing these downmodulation experiments we will be able to generate a functional map of Δ32. Subcellular localization: For intracellular co-localization studies, paraformaldehyde-fixed cells will be washed once with HBSS/BSA and then incubated in HBSS/BSA containing 0.0δ% saponin for 30 min at room temperature. Cells will be incubated with antibodies specific for endoplasmic reticulum (ER)-protein disulfide isomerase (PDI) (BDTransduction Labs, CA), golgi (golgin-97) (Molecular Probes, OR), endosomes (Rab4, δ, 7 or 11) (Becton Dickinson Biosciences.CA), lysosomeshlysosome associated membrane protein (Lamp) (Becton Dickinson Biosciences.CA), and mitochondria (Mitotracker) (Molecular Probes.OR). All immuofluorescence staining will be done at room temperature for 4δ minutes, with antibodies diluted in HBSS/BSA/0.1% saponin for 1 hour at room temperature prior to washing with PBS, drying, and mounting. Cells will be immobilized for fluorescence microscopy by mounting under poly-L-lysine coated coverslips. Samples will be viewed with a Bio- Rad MRC1024 confocal scanning laser system with a krypton/argon laser. Map the structural determinants responsible for Δ32 activity.
The data demonstrated that recombinant Δ32 caused downmodulation of not only CCRδ but also CXCR4 suggesting that it may interact with the major coreceptors using similar structural determinants. Although CCR2 shares more than 7δ% homology with CCRδ, it was not downmodulated by Δ32 suggesting specific interaction that may involve at least, part of the non-homologous region. Our data show that Δ32 expression had no significant effect on surface levels of CD4 confirming that the observed reduction in X4 fusion/infection is not due to an effect on CD4 levels (Figure 3D).
The unique carboxy-terminal 31 amino acids in the Δ32 protein create new functional domain(s) that may be specific for interaction with the major HIV-1 coreceptors. Experimental Strategy:
A direct way to determine whether the carboxy-terminal region of Δ32 is critical for its activity will be to construct chimeric molecules where this unique Δ32- specific COOH tail or portion of it is grafted onto CCRδ that lacks its carboxy tail (gain of Δ32 function). We have previously demonstrated that CCRδ lacking the carboxy terminal cytoplasmic tail is expressed at the cell surface and is functional as a coreceptor. If the biological activity of Δ32 is contained within the last 31 amino acids at its carboxy terminus, then adding this region to a tail-less CCRδ is expected to result in the loss of its ability to reach the cell surface. This analysis will be extended to include other truncation of this Δ32 fragment in order to map its interactive domain. Once the Δ32 inactivating domain is identified by chimeric constructs, further site-directed mutagenesis strategies will be employed to obtain a more detailed map of the critical amino acid residues required for Δ32-induced activity. Mutant constructs will then be used in our reporter gene activation fusion assay to study their effect on cell surface expression and coreceptor function as described in Section B.
Experimental approach: This analysis will be extended to include other truncation of this Δ32 fragment in order to map its interactive domain. Once the Δ32 inactivating domain is identified by chimeric constructs, further site-directed mutagenesis strategies will be employed to obtain a more detailed map of the critical amino acid residues required for Δ32-induced activity.
We realize that mutagenesis of the Δ32-specific 31 amino acids may not be sufficient to reveal the critical domain of this protein, however, since these amino acids are required for the observed CXCR4 downmodulation, we hypothesized that the activity of Δ32 is most probably caused by these 31 amino acids. Depending on the data we obtain using the first group of mutants that target the Δ32-specific segment, we will design mutants to include the shared homologous region with CCRδ. To gain further insight into the mechanism of Δ32 protective effect, a number of questions outside the scope of this proposal will be addressed. First, mutational analysis will be performed to determine the critical amino acid residues (coreceptor residues) involved in Δ32 interaction with the major coreceptors. Knowledge of these residues will contribute to our understanding of coreceptor structure-function and allow the design of drugs that may have a broad antiviral effect. Second, the Δ 32 may be used as a therapeutic approach to induce resistance to HIV-1 infection. Gene delivery of the Δ32 to stem cells may provide the opportunity of producing progeny cells that resist HIV-1 infection. The Δ32 protein is a naturally expressed molecule and individuals.- carrying this mutation do not show any obvious hematopoietic defects or any other immunological disorders.
Table 1.
Figure imgf000057_0001
Throughout this application, various publications, including United States patents, are referenced by author and year and patents by number. Full citations for the publications are listed below. The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The invention has been described in an illustrative manner, and it is to be understood that the terminology, which has been used is intended to be in the nature of words of description rather than of limitation.
Obviously, many modifications and variations of the present invention are , possible in light of the above teachings. It is, therefore, to be understood that within the scope of the described invention, the invention can be practiced otherwise than as specifically described.
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Claims

1 . A method of protecting individuals from contracting HIV by administering to the individuals a vector comprising a sequence encoding the Δ32 mutation.
2. The method according to claim 1 , wherein said administration step is a single administration of the vector.
3. The method according to claim 1 , wherein said administration step is a series of administrations.
4. A method of protecting individuals from the transmission and progression of HIV infection by administering to the individuals a vaccine comprising a sequence encoding the Δ32 mutation.
5. The method according to claim 4, wherein said administration step is a single administration of the vector.
6. The method according to claim 4, wherein said administration step is a series of administrations.
7. A method of decreasing the amount of HIV co-receptors present on a cell surface by administering a compound comprising a sequence encoding the Δ
32 mutation and a pharmaceutically acceptable carrier.
8. The method according to claim 7, wherein said administration step is a single administration of the vector.
9. The method according to claim 7, wherein said administration step is a series of administrations.
10. A method of treating a patient with HIV by administering a compound comprising a sequence encoding the Δ32 mutation and a pharmaceutically acceptable carrier.
1 1. The method according to claim 10, wherein said administration step is a single administration of the vector.
12. The method according to claim 10, wherein said administration step is a series of administrations.
13. A compound for decreasing the amount of HIV co-receptors present on a cell surface said compound comprising a sequence encoding the Δ32 mutation and a pharmaceutically acceptable carrier.
14. The compound according to claim 13, wherein said compound is included as the active ingredient of a vaccine.
1δ. The compound according to claim 13, wherein said sequence is a naturally occurring sequence.
16. A vector containing a sequence encoding the Δ32 mutation.
17. The vector according to claim 16, wherein said compound is included as the active ingredient of a vaccine.
18. The vector according to claim 16, wherein said sequence is a naturally occurring sequence.
19. A gene therapy comprising a sequence encoding the Δ32 mutation and a pharmaceutically acceptable carrier.
20. A therapeutic target for treating HIV, said target is Δ32 mutation.
21. An assay for testing the efficacy of HIV treatment, said assay comprising detecting means for detecting the presence of a Δ32 mutation in cells.
22. The assay according to claim 21 , wherein said detection means is PCR.
23. A vaccine for use in preventing the transmission and progression of HIV infection, said vaccine comprising a sequence encoding the Δ32 mutation and a pharmaceutically acceptable carrier.
24. The vaccine according to claim 23, wherein said sequence is a naturally occurring sequence.
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Title
MCKENZIE ET AL.: 'Nucleic acid vaccines' IMMUNOLOGIE RESEARCH vol. 24, no. 3, 2001, pages 225 - 244, XP002968550 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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