WO2009117661A2 - Carrier neutralization assay - Google Patents

Carrier neutralization assay Download PDF

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Publication number
WO2009117661A2
WO2009117661A2 PCT/US2009/037811 US2009037811W WO2009117661A2 WO 2009117661 A2 WO2009117661 A2 WO 2009117661A2 US 2009037811 W US2009037811 W US 2009037811W WO 2009117661 A2 WO2009117661 A2 WO 2009117661A2
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hiv
assay
cells
carrier
antibody
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PCT/US2009/037811
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French (fr)
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WO2009117661A3 (en
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Carl Alving
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United States Department Of The Army, As Represented By The Secretary Of The Army, On Behalf Of The Walter Reed Army Institute Of Research
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Publication of WO2009117661A2 publication Critical patent/WO2009117661A2/en
Publication of WO2009117661A3 publication Critical patent/WO2009117661A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70514—CD4

Definitions

  • This invention relates generally to a virus neutralization assay, and specifically to a neutralizing antibody carrier assay wherein said carrier may, by way of nonlimiting example, be a cell with a lipid bilayer operable to bind with HIV-1 but, itself, incapable of infecting a target cell.
  • Viruses must replicate intracellular ⁇ and often employ host cell enzymes, macromolecules, and organelles for the synthesis of virus particles. Therefore, safe and effective anti-viral compounds must be able to discriminate with a high degree of efficiency between cellular and virus-specific functions.
  • evaluation of the in vitro sensitivity of virus isolates to antiviral compounds must be carried out in a complex culture system consisting of living cells (e.g.
  • tissue culture The results from such assay systems vary widely according to the type of tissue culture cells which are employed and the conditions of assay. Viral drug resistance is a substantial problem given the high rate of viral replication and mutation frequencies. No disease better exemplifies the problem of viral drug resistance than AIDS.
  • HIV human immunodeficiency virus
  • HIV-1 is a spherical, enveloped RNA retrovirus, a lentivirus, that fuses with the plasma membrane of a host cell to insert its genomic RNA.
  • the envelope of HIV-I contains a lipid bilayer that is associated with two loosely bound glycoproteins, gp120 and gp41. These proteins are created during intracellular virus assembly when a precursor protein, gp160, is cleaved to from pg120 and gp41 (see FIG. 1A).
  • the gp41 is a trimeric transmembrane protein that is anchored in the lipid bilayer, and during viral maturation and budding the intraviral end of gp41 is bound to sites located on an N- terminal myristoylated matrix protein (p17) [1 -3].
  • HIV-1 has a diploid genome having two identical RNA molecules.
  • the molecular organization of HIV is (5 1 ) U3-R-U5-gag-pol-env-U3-R-U5 (3 1 ).
  • the U3, R, and U5 sequences form the long terminal repeats (LTR) which are the regulatory elements that promote the expression of the viral genes and sometimes nearby cellular genes in infected hosts.
  • LTR long terminal repeats
  • Gag codes for a polyprotein precursor that is cleaved by a viral protease into three or four structural proteins; pol codes for reverse transcriptase (RT) and the viral protease and integrase; env codes for the transmembrane and outer glycoprotein of the virus.
  • the gag and pol genes are expressed as a genomic RNA while the env gene is expressed as a spliced subgenomic RNA.
  • the env gene there are other HIV genes produced by spliced subgenomic RNAs that contribute to the replication and biologic activities of the virus.
  • genes include: tat which encodes a protein that activates the expression of viral and some cellular genes; rev which encodes a protein that promotes the expression of unspliced or single-spliced viral mRNAs; nef which encodes a myhstylated protein that appears to modulate viral production under certain conditions; vif which encodes a protein that affects the ability of virus particles to infect target cells but does not appear to affect viral expression or transmission by cell-to-cell contact; vpr which encodes a virion-associated protein; and vpu which encodes a protein that appears to promote the extracellular release of viral particles.
  • the human immunodeficency virus type 1 (HIV-1 ) has a lipid bilayer that contains several glycoproteins that are anchored in, or closely associated with, the membrane surface.
  • the envelope proteins have complex interactions with the lipids both on the host cells and on the target cells.
  • the processes of budding from host cells and entry into target cells occur at sites on the plasma membrane, known as lipid rafts, that represent specialized regions that are rich in cholesterol and sphingolipids.
  • the lipid_composition of the virus is largely reflective of the composition of lipid rafts present in the plasma membrane of the host cell [4, 5]. Structure, function, and lipid composition of HIV-1 and lipid interactions of HIV-1 with cells [6].
  • HIV-1 also uses lipid raft- colocalized CD4 and chemokine receptors for productive entry into CD4+ cells [13].
  • Lipid raft glycosphingolipids that have relevance to HIV -1 budding or entry include, among others, gangliosides GM 1 and GM3, GalCer, SGalCer, LacCer, and CTH (see FIG. 2).
  • HIV-1 virus After budding from host cells, the HIV-1 virus exhibits a strong tendency to infect T lymphocytes as target cells, using CD4 as a receptor [14].
  • the binding and fusion of HIV with the target cell involves a choreographed ballet between the proteins of the free virus and the entry site of the target cell (FIG. 1 B).
  • HIV entry into a cell is a multistep process initially involving the interactions of viral envelope protein gp120 and gp41 with several binding sites on the cell surface.
  • the envelope proteins exist as a trimer consisting of gp120 molecules and 3 gp41 molecules.
  • the binding of gp120 to CD4 is followed by conformational changes in the gp120 protein that expose binding sites to chemokine receptors, CXCR4 or CCR5, that serve as co-receptor binding sites for interactions of the virus with the target cell [15-17].
  • the binding of gp120 to the chemokine co-receptor induces conformational changes that allow the binding of the gp41 anchor protein to the cell, and this is followed by fusion of the viral lipid bilayer with the plasma membrane bilayer, and entry of the virion RNA into the target cell [18] (see FIG. 1 C).
  • the binding and entry processes entail numerous types interactions between proteins and lipids of the virus and specific lipids of the target cell [19]. Roles of Glycosphingolipids In HIV-1 Interactions With Host Cells
  • glycosphingolipids including GalCer and SGalCer have been proposed as alternate receptors or binding sites for HIV-1 [20-25], using a truncated form of gp120, proposed that the binding site for GalCer/SGalCer is located between amino acids 206-275.
  • a binding site of HIV-1 to GalCer has also been located to residues 650-685 of gp41 , and more specifically to residues 662-667 (comprising ELDKWA) on gp41 which is proposed to serve as a lectin for galactose-specific binding of HIV-1 to epithelial cells [20].
  • GM3 and CTH have also been indicated as binding sites for HIV-1 envelope [26-28].
  • V3 loop on gp120 has been reported to have a binding site for GalCer [29], and gp120 also binds GM3 and CTH [26-28].
  • gp120 and gp41 might both bind to GalCer, SGalCer, GM3, and CTH. It is conceivable that this could represent a sequential interaction of gp120 with the lipid bilayer, followed by interaction of gp41 with the lipid bilayer during a fusion process. This is suggested by the binding of GM3 to both the V3 loop and to CD4 [27].
  • the GM3 binding to V3 involves the sequence GPGRAF, while GM3 binds CD4 through a different sequence [27].
  • GM3 facilitates the interaction of the V3 loop with CD4 [27]. It should also be noted that the precise pg120 binding site for every glycolipid has not necessarily been delineated, and it is possible that sites other than GPGRAF might be involved in the binding of certain glycolipid headgroups. Vaccine Strategies for Developing Antibodies That would Be Effective For Blocking HIV-1
  • HIV-1 Most effective viral vaccines work, at least in part, by generating antibodies that inactivate or neutralize the invading virus, and the existing data strongly suggest that an optimally effective HIV-1 vaccine should elicit potent antiviral neutralizing antibodies.
  • HIV-1 unlike acute viral pathogens, HIV-1 chronically replicates in the host and evades the antibody response. This immune evasion, along with the large genetic variation among HIV-1 strains worldwide, has posed major obstacles to vaccine development. Current HIV vaccine candidates do not elicit neutralizing antibodies against most circulating virus strains, and thus the induction of a protective antibody response remains a major priority for HIV-1 vaccine development.
  • HIV-1 For an antibody- based HIV-1 vaccine, progress in vaccine design is generally gauged by in vitro assays that measure the ability of vaccine-induced antibodies to neutralize a broad spectrum of viral isolates representing the major genetic subtypes (clades) of HIV-1 [42].
  • virus envelope (Env) proteins have evolved an extraordinary ability to evade neutralizing antibodies, a vaccine that can elicit protective antibodies remains the best hope for developing an HIV vaccine that confers sterilizing immunity [43].
  • the main targets for neutralizing antibodies to HIV-1 are the surface gp120 and trans-membrane gp41 envelope glycoproteins (Env) that mediate receptor and coreceptor binding and the subsequent membrane fusion events that allow the virus to gain entry into cells [44].
  • Antibodies neutralize the virus by binding these viral spikes and blocking virus entry into susceptible cells, such as CD4+ T cells [45, 46].
  • susceptible cells such as CD4+ T cells [45, 46].
  • the virus exploits several mechanisms to shield itself against antibody recognition, including a dense outer coating of sugar molecules (N- linked glycans) and the strategic positioning of cysteine-cysteine loop structures on the gp120 molecule [47].
  • Induction of an effective neutralizing antibody response will require that a vaccine deliver to the na ⁇ ve B cell repertoire epitopes that are both immunogenic (i.e., possess favorable properties for B cell inductive pathways) and antigenic (i.e., available for high affinity antibody binding on functional Env spikes).
  • Viral epitopes that are conserved among most viral strains are more likely to generate cross-reactive antibodies.
  • researchers have focused on a small number of human MAbs, from clade B HIV-1 -infected individuals, that possess broadly cross-reactive neutralizing activity [48]. The cognate viral epitopes for these MAbs have been well characterized and are being evaluated as vaccine immunogens.
  • the phenomenon of virus neutralization is a function of three variables: the antibody (Ab), the virus and the target cell. Variation in any one of these parameters may drastically affect the results of assays for neutralization [49].
  • Antibody (Ab) mediated neutralization of virus particles renders virions noninfectious for permissive cells. This process of neutralization can be indirect or direct. Indirect neutralization requires secondary factors such as complement to lyse the virion, or cells which interact with the Ab-virus complex, leading, in some cases, to destruction of the virion. Direct neutralization, on the other hand, does not destroy the virus but leads to a loss of infectivity [50].
  • Abs may aggregate virus particles, rendering them non-infectious, or, with some Abs at saturating conditions, neutralization may occur by preventing virion binding to cells. But many polyclonal and monoclonal antibodies (mAbs) that neutralize do so at a post-binding step.
  • mAbs polyclonal and monoclonal antibodies
  • the critical role of Ab specificity is perhaps best illustrated by the fact that (a) an Ab that does not bind to a virus cannot neutralize it, and (b) not all Abs which bind to virions neutralize them.
  • Variation in the virus can, predictably, cause profound changes in the neutralization assay. Both the integrity of the virus preparation [51] as well as the nature of the cells used to produce the virus [52] affect the outcome of neutralization. [0022] In order to adequately monitor neutralization breadth and potency and to compare and prioritize immunogens, assays are needed that are sensitive, quantitative, high throughput, and have correlative value. The challenge is to develop standardized in vitro assays that will allow for a meaningful comparison of the quality and potency of neutralizing antibodies in sera or other fluids from HIV-positive patients and vaccine recipients. Importantly, the neutralization assay outcome may be determined by numerous assay parameters.
  • variable parameters include: target cell used and cell density, host cell used for viral stock propagation, virus dose and antibody dilution/concentration (virus particle: antibody ratio), the inclusion of complement, volumes of components added, duration of pre-incubation of virus and antibody, duration of infection with or without antibody, cell washing steps to remove unbound antibody and virus, length of culture time, the endpoint measured and other variables.
  • Neutralization platforms have been evolving over two decades. In the majority of neutralization assays, virus and antibody are incubated together and then added to CD4+ target cells. The critical concern regarding the use of any cell line-based model system is its physiologic relevance and value as a surrogate for in vivo outcomes. Subsequently, assays were developed to infect HIV seronegative peripheral blood mononuclear cells (PBMC) from healthy human donors to address these concerns. However PBMC from different donors display differential susceptibility to HIV-1 infection.
  • PBMC peripheral blood mononuclear cells
  • HIV replication may be due, in part, to host genetics and CD8+ cell factors, the number of CD4+ cells or expression levels of CD4 molecules on host cells, effects of host cell-derived molecules on the viral surface, and host genetic polymorphisms in chemokines or chemokine receptors, such as CCR5, which function as HIV-1 coreceptors. Because of these variables, the inter-lab, and even the inter- experiment variation within a single lab, is often quite problematic in PBMC-based neutralization assays.
  • the pseudoviruses are generated in the 293T cell line by cotransfection of an env-mutated viral backbone, along with the env clone of choice, and then used to infect a transformed cell line expressing the appropriate receptors.
  • One such format is an assay that employs an epithelial HeLa-dehved cell line (TZM-bl) that carries the luciferase reporter gene sensitive to the presence of the HIV Tat protein.
  • This TZM-bl assay has been advanced as a readily transferable method for assessing vaccine- elicited neutralizing antibodies in the good clinical laboratory practices (GCLP) environment.
  • Table A shows a comparison of several of the variable parameters (as described above) that distinguish the PBMC assay from the pseudovirus approach [53].
  • the pseudoviral system has several advantages as a platform for application to front-line assessment of antibodies, to include: the ability to rapidly test for neutralization against primary patient Envs from multiple clades, a high degree of inter- experiment reproducibility and throughput, ease and safety of reagent distribution for the assay, and facilitation of assay validation and global transfer.
  • specific advantages include: the ability to rapidly test for neutralization against primary patient Envs from multiple clades, a high degree of inter- experiment reproducibility and throughput, ease and safety of reagent distribution for the assay, and facilitation of assay validation and global transfer.
  • Assays employing primary cells typically capture all stages of the virus life cycle during neutralization assays incorporating multiple rounds of infection.
  • bl assay is a single round assay and primarily assesses inhibition of virus binding and entry. It is possible that some antibody subpopulations may not be detected through the use of a single assay focusing only on virus entry.
  • Another fundamental difference between T cells and the HeLa-derived TZM-bl cells may lie in the location of HIV/receptor interaction(s). The majority of HIV-1 entry into T cells occurs at the plasma membrane via CD4 and chemokine receptor-mediated engagement and fusion. In HeLa cells, endocytosis has been reported to account for approximately 85% of virus entry [54].
  • TZM-bl assays often include the use of DEAE-dextran during the infection phase. If endocytosis is playing a significant role in viral entry in TZM-bl cells, this compound most likely enhances productive infection by buffering endosomes, thus allowing the pseudovirus to avoid lysosomal degradation and to enter the cell through
  • antigenic epitopes i.e., molecular recognition sites for binding of antibodies
  • the antibodies are reacting directly with the free virus; or with the envelope antigens as they undergo conformational changes during the entry process into target cells; or with the target cells themselves; or with the budding virus as it emerges from the host cell; or with the host cell itself; or even whether the antibody actually enters the target or host cell (e.g., through endocytosis or pinocytosis) where it might interact with the intracellular virus that is maturing within the cell.
  • What is needed is an assay that allows direct interaction of potentially neutralizing antibodies with HIV-1 itself in the absence of host cell or target cell interactions, but which allows subsequent assay of the ability of the HIV-1 that has been exposed to antibody, for the ability (or inability) to infect a target cell.
  • the present invention provides such an assay.
  • a method of performing an assay comprising the steps of: incubating a carrier with a said test sample to produce a first complex; incubating an analyte with said first complex to produce a second complex ; and, performing a measurement on said complex.
  • FIG. 1 is a diagram of the interactions of HIV-1 envelope proteins with plasma membrane lipids during target cell binding (B) and fusion steps (C);
  • FIG. 2 is a structural drawing of ceramide and selected gylcosphingolipidsthat bind and interact with HIV-1 envelope proteins, or with CD4 or chemokine receptors, during binding and fusion of HIV-1 with target cells.
  • FIG. 3 is a table showing key details of various neutralization assay protocols.
  • FIG. 4 is a table showing key differences between neutralization assay protocols.
  • FIG. 5 is a diagram of the plasma membrane glycosphingolipid microdomains as preferential sites of formation of the HIV-1 fusion complex
  • FIG. 6 is a schematic diagram of HIV-1 gp41 envelope protein
  • FIG. 7 is a model of the HIV-1 molecule showing gp41 at the vicinity of the lipid bilayer.
  • FIG 8 is line graph showing percent neutralization of the carrier-neutralization assay as a function of antibody saturation.
  • adjuvant An adjuvant is defined as anything that will amplify the immune response or improve the immune response over what the immune response would be without the adjuvant.
  • analyte is the substance to be detected which may be present in the test sample.
  • the analyte can be any substance for which there exists a naturally occurring specific binding member (such as, an antibody), or for which a specific binding member can be prepared.
  • an analyte is a substance that can bind to one or more specific binding members in an assay.
  • “Analyte” also includes any antigenic substances, haptens, antibodies, and combinations thereof.
  • the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of intrinsic factor protein as a member of a specific binding pair for the determination of Vitamin B12, the use of folate-binding protein to determine folic acid, or the use of a lectin as a member of a specific binding pair for the determination of a carbohydrate.
  • the analyte can include a protein, a peptide, an amino acid, a nucleotide target, and the like.
  • an "antigenic epitope” is a molecular recognition site for binding of antibodies. Commonly this is determined or produced by injecting an antigenic material into a mammal, or by introduction of the antigenic material to lymphocytes in vitro, for presentation of the antigenic material to lymphocytes to induce antibodies that are secreted by lymphocytes, and said antibodies then have the capacity to bind to sites on the material that had been presented to the lymphocytes.
  • Broadly neutralizing A commonly encountered problem in HIV-1 immunology and vaccinology is the inability of antibodies induced against HIV-1 organisms produced in the laboratory to prevent (i.e., neutralize) primary isolates of HIV-1 viruses from infecting target cells. Broadly neutralizing antibodies are defined as antibodies that have the ability to partially or completely overcome this problem by neutralizing more than one type of primary isolate of HIV-1 virus.
  • a “Carrier” is a nontoxic, cell, cellular component or molecule to which a virus, pseudovirus and/or viral subunits are conjugated by physiological or biochemical means.
  • Carrier bound HIV-1 a carrier as defined herein bound to HIV-1 by physiological or biochemical means.
  • a "Candidate Neutralizing Anitbody” means broadly neutralizing HIV antibodies such as, by way of nonlimiting example, 4E10, 2F5, and Z13: These are designations of monoclonal antibodies, derived from individual humans infected with HIV-1 , or that have been identified from phage display libraries, that have the ability to broadly neutralize clinical isolates of HIV-1. The antibodies are further taught and described by Buchacher et al. [Buchacher A, Predi R, Strutzenberger K, Steinfellner W, Trkola A, Purtscher M, Gruber G, Tauer C, Steindl F, Jungbauer A, Katinger H.
  • Dual-specific or multi-specific This is defined as the ability of an antibody to bind simultaneously or independently to epitopes on two or more types of antigenic chemical species, for example to an amino acid sequence and to a lipid; or to a sugar and a lipid; or to an amino acid sequence and a sugar.
  • the term “dual” refers only to binding to more than one type of chemical epitope, but such antibody binding specificities may actually contain as many molecular binding sites for different types of chemical epitopes (including three, or more, epitopes) as there is available space on the binding site of the antibody for such simultaneous binding of more than one type of epitope.
  • enveloped virus A virus that has an envelope (i.e., an outer lipid bilayer structure together with associated proteins on the outer surface) is an enveloped virus.
  • enveloped virus examples include: HIV-1 , influenza virus, dengue virus, Sindbis virus, and Ebola virus, among many others.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • ELISA is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample.
  • the ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.
  • an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen.
  • This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal.
  • fluorescence ELISA when light of the appropriate wavelength is shown upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.
  • Performing an ELISA involves at least one antibody with specificity for a particular antigen.
  • the sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA).
  • a solid support usually a polystyrene microtiter plate
  • the detection antibody is added, forming a complex with the antigen.
  • the detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation.
  • the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound.
  • the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.
  • Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates enabling much higher sensitivity.
  • the ELISA test or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.
  • EIA enzyme immunoassay
  • a person's serum is diluted 400-fold and applied to a plate to which HIV antigens have been attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum.
  • a specially prepared "secondary antibody” an antibody that binds to other antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate.
  • a concentrated solution of non-interacting protein such as bovine serum albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.
  • BSA bovine serum albumin
  • the plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
  • the plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
  • Secondary antibodies which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
  • the enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules.
  • a major disadvantage of the indirect ELISA is that the method of antigen immobilization is nonspecific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface.
  • the sandwich ELISA provides a solution to this problem.
  • ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.
  • Sandwich ELISA A less-common variant of this technique, called “sandwich” ELISA, is used to detect sample antigen. The steps are as follows:
  • Unlabeled antibody is incubated in the presence of its antigen.
  • the secondary antibody specific to the primary antibody is added. This second antibody is coupled to the enzyme. [0085] A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
  • ELISA Reverse method & device (ELISA-R m&d).
  • a newer technique uses a solid phase made up of an immunosorbent polystyrene rod with 4-12 protruding ogives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents.
  • the ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and different antigens for multi-target assays;
  • the sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples;
  • One ogive is left unsensitized to measure the non-specific reactions of the sample;
  • the use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating ready-to-use lab-kits and on-site kits.
  • Flow cytometric procedures and particle counting procedures include processes whereby analytes which are antibody members of specific binding pairs are quantified by mixing an aliquot of test sample suspected of containing a specific antibody with microparticles coated with a capture reagent specific for such antibody such as at least one of the peptides disclosed herein, capable of binding to the antibody of interest as the other member of the specific binding pair. If the antibody is present in the test sample, it will bind to some of the microparticles coated with the capture reagent and agglutinates will form. The analyte concentration is inversely proportional to the unagglutinated particle count. See, for example, Rose et al., eds., Manual of Clinical Laboratory Immunology, 3rd edition, Chapter 8, pages 43-48, American Society for Microbiology, Washington, D.C. (1986).
  • Flow cytometry methods that sense electronic and optical signals from cells or particles which are illuminated allows determination of cell surface characteristics, volume and cell size.
  • Antibody present in, for example, a test sample are bound to a peptide disclosed herein and detected with a fluorescent dye which is either directly conjugated to the peptide or added via a second reaction.
  • Different dyes which may be excitable at different wavelengths, can be used with more than one peptide specific to different analytes such that more than one analyte can be detected from one sample.
  • fluorescence flow cytometry a suspension of particles, typically cells in a test sample, is transported through a flowcell where the individual particles in the sample are illuminated with one or more focused light beams.
  • One or more detectors detect the interaction between the light beam(s) and the labeled particles flowing through the flowcell. Comnnonly, some of the detectors are designed to measure fluorescence emissions, while other detectors measure scatter intensity or pulse duration. Thus, each particle that passes through the flowcell can be mapped into a feature space whose axes are the emission colors, light intensities, or other properties, i.e., scatter, measured by the detectors. In one situation, the different particles in the sample map into distinct and non-overlapping regions of the feature space, allowing each particle to be analyzed based on its mapping in the feature space. To prepare a test sample for flow cytometry analysis, the operator manually pipettes a volume of test sample from the sample tube into an analysis tube.
  • a volume of the desired fluorochrome labeled peptide is added.
  • the sample/peptide mixture then is incubated for a time and under conditions sufficient to allow antibody/peptide bindings to take place. After incubation, and if necessary, the operator adds a volume of RNS lyse to destroy any RBCs in the sample. After lysis, the sample is centhfuged and washed to remove any left-over debris from the lysing step. The centrifuge/wash step may be repeated several times. The sample is resuspended in a volume of a fixative and the sample then passes through the fluorescence flow cytometry instrument.
  • a "hapten” is a small molecule which can elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one which also does not elicit an immune response by itself.
  • the term "hapten”, as used herein, refers to a partial antigen or non-protein binding member which is capable of binding to an antibody, but which is not capable of eliciting antibody formation unless coupled to a carrier protein. Haptens may be used to enhance the signal generated, and thus the sensitivity of the assay. The use of haptens is known in the art. It is contemplated that haptens also can be used in assays employing the peptides disclosed herein in order to enhance performance of the assay.
  • the "indicator reagent” comprises a “signal generating compound” (label) which is capable of generating and generates a measurable signal detectable by external means conjugated (attached) to a specific binding member for HIV.
  • label a “signal generating compound” as used herein means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule.
  • the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like.
  • An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to HIV as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
  • Lipids are defined as taught by Small, D. M., “The Physical Chemistry of Lipids, From Alkanes to Phospholipids” Handbook of Lipid Research, VoI, 4, Plenum, NY, 1986, p. 1 , as given below:
  • lipids Assuming a broad definition, one can define a lipid as any molecule of intermediate molecular weight (between 100 and 5000) that contains a substantial portion of aliphatic or aromatic hydrocarbon. Included are the hydrocarbons, steroids, soaps, detergents, and more complex molecules, such as triacylglycerols, phospholipids, gangliosides, and lipopolysaccharides. Immediately, one can imagine that the physical behavior of such chemically divergent molecules will be quite different.
  • lipids are their tremendously varied behavior in aqueous systems, ranging from almost total insolubility (e.g., paraffin oil and sterol esters) to nearly complete solubility (e.g., soaps, detergents, bile salts, and gangliosides). This particular aspect of lipids is important biologically because all cells exist in an aqueous milieu.”
  • insolubility e.g., paraffin oil and sterol esters
  • solubility e.g., soaps, detergents, bile salts, and gangliosides
  • Lipid structure (this includes all organized lipid structures, or domains, and all solid phase, mesomorphic, crystalline, liquid crystalline, and liquid lipid structures): This is defined as all of the multiple organized physical states of lipids, as taught by Small, D. M., in “The physical states of lipids: solids, mesomorphic states, and liquids” in “The Physical Chemistry of Lipids, From Alkanes to Phospholipids" Handbook of Lipid Research, VoI, 4, Plenum, NY, 1986, Chapter 3, pp. 43-87. All of the above terms are interchangeable as defined in the context of this invention.
  • solid phase lipid structure is interchangeable with “mesomorphic states”, “liquid lipids”, “organized lipid structures” “domains”, “crystalline lipid structures”, liquid crystal lipid structures”, and “liquid lipid structures”.
  • Lipid bilayer membrane This is a type of double layer membrane in which the polar groups of the parallel array of lipids of each monolayer of lipids are oriented toward the aqueous phase and the nonpolar groups (such as fatty acyl groups) of each monolayer are oriented toward each other in the center of the bilayer. Liposomes often contain lipid bilayers, as do plasma membranes of cells.
  • Liposomes as they are ordinarily used, consist of smectic mesophases, and may consist or either phospholipid or nonphospholipid smectic mesophases.
  • a “neutralization assays” is an assay format designed to utilize the carrier complexes described herein which contain antigenic epitopes that are useful in competitive assays such as described herein.
  • a neutralization assay a peptide representing an epitope of an antigenic region of HIV-1 , is solubilized and mixed with a sample diluent to a final concentration of between 0.5 to 50.0 . ⁇ g/ml.
  • a known amount of test sample for example, 10 ⁇ l
  • either diluted or non- diluted is added to a reaction well, followed by, for example, 400 . ⁇ l of the sample diluent containing a peptide (such as the carrier complex described herein).
  • the mixture may be preincubated for approximately 15 minutes to two hours.
  • a solid phase coated with the peptide is then is added to the reaction well, and incubated for one hour at approximately 40 degrees C.
  • a known amount of an indicator reagent for example, 200 . ⁇ l of a peroxidase labelled goat anti-human IgG in a conjugate diluent, is added and incubated for about one hour at 40 degrees C.
  • an enzyme substrate for example, OPD substrate, is added to the mixture and incubated at room temperature for thirty minutes.
  • the reaction is terminated by adding a stopping reagent such as 1 N sulfuric acid to the reaction well. Absorbance is read at 492 nm.
  • Test samples which contain antibody to the specific peptide generate a reduced signal caused by the competitive binding of the peptides to these antibodies in solution.
  • the percentage of competitive binding may be calculated by comparing absorbance value of the sample in the presence of peptide to the absorbance value of the sample assayed in the absence of a peptide at the same dilution.
  • the difference in the signals generated between the sample in the presence of peptide and the test sample in the absence of peptide is the measurement used to determine the presence or absence of antibody.
  • Opsonization is the process by which a pathogen is marked for ingestion and destruction by a phagocyte.
  • Opsonization involves the binding of an opsonin, i.e., antibody, to a receptor on the pathogen's cell membrane. After opsonin binds to the membrane, phagocytes are attracted to the pathogen.
  • the Fab portion of the antibody binds to the antigen, whereas the Fc portion of the antibody binds to an Fc receptor on the phagocyte, facilitating phagocytosis.
  • the receptor-opsin complex can also create byproducts like C3b andC4b which are important components of the complement system. These components are deposited on the cell surface of the pathogen and aid in its destruction.
  • the cell can also be destroyed by a process called antibody-dependent cellular cytotoxicity in which the pathogen does not need to be phagocytosed to be destroyed. During this process, the pathogen is opsonized and bound with the antibody IgG.
  • the antibody triggers a release of lysis products from cells like monocytes, neutrophils, eosinophils, and natural killer cells. This process can cause inflammation of surrounding tissues and damage to healthy cells. (Antibody Opsonization)
  • P24 Antigen Assay Viral isolation through viral culture, nucleic acid tests to detect viral RNA, and tests to detect p24 antigen can be used to demonstrate virus or viral components in blood, thereby verifying infection.
  • the p24 antigen assay measures the viral capsid (core) p24 protein in blood that is detectable earlier than HIV antibody during acute infection.
  • p24 antigen is found in serum in either free form or bound by anti-p24 antibody. Free p24 can be measured with enzyme immunoassays whereas detection of bound p24 requires pretreatment with an acid to dissociate the complex.
  • HIV p24 antigen tests employ ELISA technology with modifications to detect antigen, not antibody.
  • a specific monoclonal antibody to HIV p24 is attached to the solid phase (microtiter plate-well or polystyrene bead) acting to "capture” the viral antigen in the sample when added.
  • the sample is diluted in a Triton X100 detergent to disrupt virions, and if antigen is present in the serum, the antigen will attach to the monoclonal antibody on the solid phase.
  • an antibody detector is added and incubated. This detector reagent is usually a high-titer antibody to p24 antigen that is coupled to biotin.
  • a conjugate streptavidin-peroxidase
  • An avidin-biotin system acts as an amplifier to generate additional signal to detect the small quantities of antigen in the sample.
  • Addition of a substrate tetramethylbenzidine
  • a weak acid e.g., 2 M sulfuric
  • Resultant optical density values are proportional to the amount of HIV-1 p24 antigen in the specimen. This assay can detect p24 antigen in the pg/ml-to-ng/ml range.
  • the optical density is read with a spectrophotometer at 450 nm.
  • a protocol for an in-house p24 antigen assay designed for testing large numbers has been described and is more cost effective than commercially available assays. (27 Mckeating J. Quantitative assays for virus neutralization. In: Karn J, ed. HIV: A Practical Approach, Virology and Immunology, vol 1. Oxford: IRL Press at Oxford University Press, 1995;118-127.)
  • an HIV-1 antigen standard is diluted to prepare a series of six standards of varying concentrations. Concentrations vary between 0.0 and 125 pg/ml. A standard curve is generated from which optical density values of the unknown specimens are interpolated to determine their concentration. The standard curve is constructed using a linear graph and plotting the concentration of the HIV-p24 antigen standard (pg/ml) on the X-axis versus the mean optical densities for each standard on the Y-axis. Each standard is added in duplicate wells, and at least 5 controls must be included (3 negatives and 2 positives). If the value of the unknown sample is higher than the value of the highest standard, the sample must be diluted in normal human serum and the entire neutralization procedure is repeated.
  • an Immune Complex Dissociation (ICD) procedure may be introduced using low pH to dissociate p24 antigen/anti-p24antibody complexes before performing the antigen assay.
  • ICD Immune Complex Dissociation
  • This dissociation procedure allows for detection of both free p24 antigen and complexed p24 antigen/antibody.
  • the method not only increases the number of antigen positive individuals (epidemiologic sensitivity), but also can detect lower amounts of p24 antigen (analytical sensitivity).
  • the sensitivity of this assay may be further enhanced using a signal amplifying step that involves the addition of a tyramide compound which generates an intermediate to produce more enzyme and substrate molecules; and hence, more signal.
  • a "Peripheral Blood Mononuclear Cell” is a blood cell having a round nucleus, such as a lymphocyte or a monocyte. These blood cells are a critical component in the immune system to fight infection and adapt to intruders.
  • the lymphocyte population consists of T cells (CD4 and CD8 positive -75%), B cells and NK cells (-25% combined). These cells are often extracted from whole blood using ficoll, a hydrophilic polysaccharide that separates layers of blood, with monocytes and lymphocytes forming a buffy coat under a layer of plasma. This buffy coat contains the PBMCs.
  • PBMC can be extracted from whole blood using a hypotonic lysis which will preferentially lyse red blood cells. This method results in neutrophils and other polymorphonuclear (PMN) cells which are important in innate immune defense to be obtained.
  • PMN polymorphonuclear
  • PBMCs are widely used in research and clinical uses every day. HIV research uses them because PBMCs include CD4+ cells, the cells HIV infects.
  • PBMC Peripheral Blood Mononuclear Cells Assay PBMC were prepared by Percoll centrifugation and depleted of CD8 cells with anti-CD8 antibody-coated magnetic beads (Dynabeads, DYNAL). Virus isolation was carried out by coculture of PBMC from infected and normal individuals after stimulation with anti-CD3 antibody (clone CLB-CD3). PBMC grown for 4 days were infected with the virus inoculum and incubated for 3 days in culture medium containing serially decreasing concentrations of antiretroviral drugs.
  • the p24 concentrations in culture supernatant were measured with mini VIDAS (bioMerieux) and the 50% and 90% inhibition concentrations (IC50 and IC90) were obtained from the dose-response curve.
  • the antiretroviral drugs being assayed were AZT, ddl, 3TC, d4T, ABC, NVP, EFV, IDV, SQV, NFV, APV, and LPV.
  • the HIV-1 LAI isolate was used as reference.
  • the protocol is a two-step procedure that first involves cocultivation of patient infected peripheral blood mononuclear cells (PBMC) with seirnegative phytohemagglutinin-stimulated donor PBMC to obtain an HIV-1 stock.
  • the virus stock is titrated for viral infectivity (50% tissue culture infective dose) by use of serial fourfold virus dilutions in donor PBMC.
  • a standardized inoculum of 1 ,000 50% tissue culture infective doses per 106 cells is used in the second step of the procedure to acutely infect seronegative donor PBMC in a 7-day microtiter plate assay with triplicate wells containing zidovudine (ZDV) concentrations ranging from 0 to 5.0 ⁇ M.
  • ZDV zidovudine
  • the conventional PBMC based assay [59-61] with readout based on p24 antigen production involves multiple rounds of virus replication, has a moderate reproducibility and sensitivity, is time-consuming and cumbersome to perform but involves the most physiological target cell.
  • An alternative readout can be the measurement of viral RNA, which shortens the time by several days [62].
  • Intracellular (IC) p24 antigen determination in infected PBMC cultures may be run as a single round assay with increased sensitivity, reproducibility and speed but it is not easy to perform [63].
  • the method of measuring ICp24 was also applied to other target cells, like macrophages [64].
  • Plaque reduction assays use either U87.CD4 or GHOST(3) cells engineered to express coreceptors for HIV [65].
  • U87.CD4 cells the syncytium- inducing capacity of HIV is exploited, while infected GHOST(3) cells turn green due to the activation of the GFP gene linked to the HIV-2 LTR.
  • the fusion assay is based on fusion of effector cells expressing the native HIV-1 envelope on their surface (PM 1 persistently infected with HIV-1 ) with target cells expressing the appropriate receptors (initially NIH-3T3 mouse fibroblasts or HeLa human epithelial cells stably expressing human CD4, CCR5 and/or CXCR4).
  • the readout is measurement of ⁇ -galactosidase activity [66].
  • Pseudovirus (PSV)-based assays exist in a number of variant assay formats using different target cells [67, 68]. A selected molecular clone is tested in a single round assay with luciferase readout that results in short-term assays with high reproducibility and sensitivity.
  • Primary isolates of HIV-1 are isolates of HIV-1 that are found spontaneously in human populations. Commonly, such isolates are obtained from clinical specimens taken from individuals naturally infected with HIV-1. Primary isolates differ from latoratory isolates in that the latter are strains of HIV-1 that are adapted to growth in transformed T cell lines.
  • the various "signal generating compounds" contemplated include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, achdiniums, phenanthridiniums and luminol, radioactive elements, and direct visual labels.
  • luminescent compounds such as fluorescein and rhodamine
  • chemiluminescent compounds such as dioxetanes, achdiniums, phenanthridiniums and luminol
  • radioactive elements and direct visual labels.
  • enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like.
  • the selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
  • Solid phases are known to those in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes (stabilized human red cells) and others.
  • the "solid phase” is not critical and can be selected by one skilled in the art.
  • latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples.
  • solid phase refers to any material which is insoluble, or can be made insoluble by a subsequent reaction.
  • the solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
  • the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
  • the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
  • the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction.
  • the receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay.
  • the solid phase thus can be a plastic, derivatized plastic, magnetic or nonmagnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes and other configurations known to those of ordinary skill in the art.
  • the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens.
  • Microporous structures are generally preferred, but materials with gel structure in the hydrated state may be used as well. These materials may be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics.
  • the methods of the present invention can be adapted for use in systems which utilize microparticle technology including in automated and semi-automated systems wherein the solid phase comprises a microparticle (magnetic or non-magnetic).
  • SPM scanning probe microscopy
  • the capture phase for example, at least one of the peptides disclosed herein
  • a test sample suspected of containing the antibody of interest is contacted with the solid phase and a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase.
  • scanning tunnelling microscopy eliminates the need for labels which normally must be utilized in many immunoassay systems to detect antigen/antibody complexes.
  • SPM to monitor specific binding reactions can occur in many ways.
  • one member of a specific binding partner is attached to a surface suitable for scanning.
  • the attachment of the analyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art.
  • covalent attachment of a specific binding partner (analyte specific substance) to a test piece which test piece comprises a solid phase of dehvatized plastic, metal, silicon, or glass may be utilized.
  • Covalent attachment methods are known to those skilled in the art.
  • polyelectrolyte interactions may be used to immobilize a specific binding partner on a surface of a test piece by using techniques and chemistries.
  • the surface may be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding.
  • the surface also may be scanned either at the site of manufacture or point of use to verify its suitability for assay purposes. The scanning process is not thought to alter the specific binding properties of the test piece.
  • a "specific binding member,” as used herein, is a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog.
  • Immunoreactive specific binding of folate-binding protein to determine folic acid or the use of a lectin as a member of a specific binding pair for the determination of a carbohydrate.
  • the specific binding pair member can include a protein, a peptide, an amino acid, a nucleotide target, and the like.
  • specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog.
  • Immunoreactive specific binding members include antigens, antigen fragments, antibodies and antibody fragments, both monoclonal and polyclonal, and complexes thereof, including those formed by recombinant DNA molecules.
  • test sample refers to a component of an individual's body which is the source of the analyte (such as, antibodies of interest or antigens of interest). These components are well-known in the art.
  • biological samples include biological samples which can be tested by the methods described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; and biological fluids such as cell culture supernatants; fixed tissue specimens; and fixed cell specimens.
  • FIG. 5 Plama membrane glycosphingolipid microdomains as preferential sites of formation of the HIV-1 fusion complex.
  • CD4 1 is present in glycoshingolipid enriched microdomains but is not associated with HIV-1 coreceptors.
  • the viral particle 2 is conveyed to an appropriate coreceptor 3 by the glycosphingolipid raft 4, which moves freely in the external leaflet of the plasma membrane 5, cholesterol 6; glycosphigolipid 4; phosphatidylcholine 7.
  • FIG. 5 Plama membrane glycosphingolipid microdomains as preferential sites of formation of the HIV-1 fusion complex.
  • CD4 1 is present in glycoshingolipid enriched microdomains but is not associated with HIV-1 coreceptors.
  • the viral particle 2 is conveyed to an appropriate coreceptor 3 by the glycosphingolipid raft 4, which moves freely in the external leaflet of the plasma membrane 5, cholesterol 6; glycosphigolipid 4; phosphatidylcholine 7.
  • FIG. 1A is a cross section of the HIV-1 envelope protein.
  • the binding and fusion of HIV with the target cell involves a choreographed ballet between the proteins of the free virus 2 and the entry site of the target cell (FIGS. 1 B and 1C).
  • HIV entry into a cell is a multistep process initially involving the interactions of viral envelope protein gp120 and gp41 with several binding sites on the cell surface.
  • the envelope proteins exist as a trimer consisting of 3 gp120 molecules and 3 gp41 molecules.
  • the binding of pg120 to CD3 is followed by conformational changes in the gp120 protein that expose binding sites to chemokine receptors 3, CXCR4 or CCR5, that serve as co-receptor binding sites for interactions of the virus with the target cell (Berger ct al., 1999; Doms, 2000; Huang et al 2005).
  • the binding of gp120 to the chemokine co-receptor induces conformational changes that allow the binding of the gp41 anchor protein to the cell, and this is followed by fusion of the viral lipid bilayer with the plasma membrane bilayer, and entry of the virion RNA into the target cell (Colman & Lawrence, 2003) (FIG. 1C).
  • the binding and entry processes entail numerous types interactions between proteins and lipids of the virus and specific lipids of the target cell (Fantini et al. 2002).
  • FIG. 1 the reference numbers represented as follows CD4 1 , viral particle 2, co-receptor 3, raft 4, plasma membrane 5, p17 matrix 10, lipid bilayer 11 , membrane proximal region 18, fusion peptide 19.
  • the arrow represents step 3 of fusion and entry.
  • Two such conserved antigenic regions are portions of the Membrane Proximal Region (MPR) of gp41 and the lipid bilayer itself, including lipids such as phosphatidylinositol phosphate, phosphatidylserine, phosphatidylglycerol, and cholesterol.
  • MPR Membrane Proximal Region
  • the MPR region 18 of gp41 as shown in FIG. 6 contains the binding epitopes for two human IgG monoclonal antibodies that are known to be broadly neutralizing antibodies. They are known as 2F5 and 4E10. 2F5 binds to ELDKWA (the MPR starts at D) and 4E10 binds to NWFDIT.
  • the 2F5 epitope, ELDKWA is the same sequence identified as the binding site for GalCer.
  • Both the 2F5 and 4E10 epitopes on gp41 are shown as labeled on FIG. 7.
  • the cholesterol binding site LWYIK is at the end of the MPR.
  • FIG. 7 is a schematic model of the HIV-1 putative trimehc envelope spike.
  • the viral particle 2 is shown inserted into the plasma membrane 5.
  • Most of the surface of gp41 is believed to be occluded by gp120.
  • the amino acid sequences of gp41 close to the membrane that have been identified as binding sites of MABs 2F5, Z13, and 4E10 have been suggested to be exposed to antibody binding [70].
  • IgG is shown as 20.
  • the HIV-1 virus is allowed to bind to a carrier in such a manner that the carrier-bound HIV-1 retains the ability to infect a target cell.
  • the carrier-bound HIV-1 Prior to such an infection the carrier-bound HIV-1 is exposed to candidate neutralizing antibodies.
  • the unbound candidate neutralizing antibodies are then removed by washing the carrier free of unbound antibodies.
  • a cell lacking CD4 (a CD4-negative cell) were to serve as a carrier for binding to HIV-1 , and if the CD4- negative cell could not be infected by the HIV-1 , the HIV-1 would be retained on the surface of the cell where it could be tested for binding of antibody and the unbound antibody could be washed off by a variety of standard laboratory methods, such as simple centhfugation, or other methods of separating cells from the surrounding liquid milieu.
  • HIV-1 binds to CD4-negative cells through several mechanisms.
  • One way is through mediation of antibodies binding to HIV-1 to form immune complexes, and the immune complexes binding to complement receptor type I (CR1 ) on erythrocytes.
  • CR1 complement receptor type I
  • Another mechanism is through binding of HIV- 1 to Duffy blood group antigen [73].
  • Yet another mechanism is through direct binding of HIV-1 to CD4-negative cells in the circulation [74].
  • HIV-1 that is bound to CD4-negative cells such as Raji cells (B lymphocytes), erythrocytes, platelets, neutrophiles, etc.
  • CD4-negative cells such as Raji cells (B lymphocytes), erythrocytes, platelets, neutrophiles, etc.
  • free virus 17 times more infectious when HIV-is bound to Raji cells, and 2-3 times more infectious when HIV-1 is bound to erythrocytes or platelets.
  • the infectivity of CD4-positive cells occurred through cell-to-cell contact. Presumably, the CD4 binding had the higher affinity for HIV-1 binding, thus promoting transfer of the HIV-1 to the CD4-positive cells (see Table 1 from dinger et al., [74] incorporated by reference in its entirety herein).
  • glycosphingolipids on the cell surfaces such as GalCer, CTH, GM3, or other similar lipids that bind HIV-1 [74].
  • the glycosphingolipids might have become exposed in damaged or senescent erythrocytes, and such cells are known to express simple glycosphingolipids (such as alpha-GalCer) that are thought to allow binding of natural antibodies (such as antibodies that recognize glycosphingolipids that have a terminal alpha-Gal) for removal of senescent erythrocytes [75].
  • CTH is an example of a glycolipid with a terminal alpha galactose.
  • Lipid bilayer lipids are cryptic antigens in normal cells, and are protected from binding of antibodies to lipids by abundant overlying membrane proteins or glycoproteins. This is dramatically illustrated by the observation that large numbers of B cells that secrete IgM or IgG antibodies to phosphatidylcholine are easily demonstrated in mice, rats, rabbits, and humans [76-78]. These antibodies are capable of binding to phosphatidylcholine, or to liposomes containing [79, 80], but binding to phosphatidylcholine on erythrocytes occurs only when the erythrocytes have been treated with a proteolytic enzyme (bromelin) [76-78, 80-82]. Erythrocytes that are aged have reduced amounts of sialic acid that is thought to allow removal of senescent cells [83], and this might promote increased visibility of the underlying lipids.
  • a proteolytic enzyme bromelin
  • HIV-1 that is circulating in the blood becomes bound to circulating CD4-negative elements of the blood that serve as carriers that cannot be infected (see above). Since the bound virus has greatly increased infectivity for CD4- positive T lymphocytes when compared to free virus, a new type of neutralization assay could be developed by pre-treatment of the CD4-negative cell-HIV-1 complex with presumptive neutralizing antibodies. The unbound antibodies could be removed (for example after binding to erythrocytes) by centrifugation or other types of washing, and the washed cells would be co-cultured with CD4-positive target cells (for example, peripheral blood mononuclear cells or H9 T cell line) as an indicator for whether the antibodies neutralized the carrier-bound HIV-1. For each assay run, the positive control would consist of the CD4-negative cell-HIV-1 complex that would exhibit the expected level of infectivity of co-cultured CD4-positive cells.
  • CD4-positive target cells for example, peripheral blood mononuclear cells or H9 T cell line
  • erythrocytes are used as CD4-negative carriers, the cells themselves could be examined for the effects of antibody-mediated complement activation in the presence of the bound antibodies by hemolyzing the cells in a standard complement lysis assay.
  • the Duffy antigen is a protein located on the surface of red blood cells and is named after the patient in which it was discovered. In humans, this protein is encoded by the DARC gene. The protein encoded by this gene is a glycosylated membrane protein and a non-specific receptor for several chemokines. The protein is also the receptor for the human malarial parasites Plasmodium vivax and Plasmodium knowlesi. Polymorphisms in this gene are the basis of the Duffy blood group system. The gene is also known as CCBP1 , Glycoprotein D (GPD), Dfy and CD234.
  • the Duffy Antigen Receptor for Chemokines belongs to a family of erythrocyte chemokine receptors that bind C-X-C and C-C chemokines such as interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1 ) and regulated-on- activation, normal T cell-expressed and -secreted (RANTES), but not macrophage inflammatory protein 1 alpha (MIP-1 alpha) or MIP-1 beta.
  • IL-8 interleukin 8
  • MCP-1 monocyte chemoattractant protein 1
  • RANTES normal T cell-expressed and -secreted
  • MIP-1 alpha macrophage inflammatory protein 1 alpha
  • MIP-1 beta macrophage inflammatory protein 1 beta
  • RBCs may function as a reservoir for HIV-1 or as a receptor for the entry of HIV-1 into CD4-cell subsets as well as neurons or endothelial cells. HIV-1 attaches to RBCs via DARC, effecting trans-infection of target cells.
  • Red cells were washed three times in cold PBS (pH 7.4), resuspended in the same solution, and mixed continuously overnight at 4 0 C with anti-Fy6 antibody at a concentration of 10 ⁇ g/ml of packed red cells. This concentration, determined with radioiodinated antibody, exceeds the concentration required to saturate Duffy antigen sites. Unbound antibody was removed by washing the red cells with cold PBS. Red cell ghosts were prepared by hypotonic lysis with 20 volumes of cold 5 mM sodium phosphate buffer (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride and 100 kallikrein-inactivating units/ml Trasylol (aprotinin).
  • Ghosts were washed exhaustively until they were light pink in color. ghosts were centhfuged for 30 min at 43,000 X g; supernatant was decanted, and the pellet was made to 50 mM Hepes- NaOH, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 100 kallikrein-inactivating units/ml Trasylol, and frozen at -20 0 C. Frozen ghosts prepared in this way can be stored for months without loss of Duffy antigens.
  • the detergent extract was then incubated with Sepharose 4B beads coupled to anti-mouse IgG for 1 h at room temperature.
  • the ratio of beads to detergent extract was 1 :100 (v/v).
  • the antimouse IgG-Sepharose beads were removed by centrifugation, and washed in a solution containing PBS and 0.5% Triton X-100 at a 1 :20 (v/v) ratio of beads to washing solution. The washings were done at room temperature for 15 min and repeated three times.
  • Elution was done by incubating the beads in a solution containing 62.5 mM Ths-HCL (pH 6.8), 0.5% SDS at a 1 :2 (v/v) ratio beads to eluant. The incubation was at 65 0 C for 10 min and repeated three times. The eluted material was concentrated in an Amicon concentrator with PM Y10 filter (Amicon Corp.) under nitrogen pressure.
  • First-strand cDNA was synthesized from Fy(a-b+) phenotype mRNA using the preamplification kit from BRL and oligo(dT) as primer.
  • cDNA, primer A, primer B, and Taq polymerase (Stratagene) were incubated in a Perkin-Elmer thermal DNA cycler.
  • the amplification product of expected size 72 bp was subcloned in pBluescript-SK vector (Stratagene).
  • the deduced amino acid sequence of the insert matched the sequence of Pe-5 peptide.
  • oligonucleotide TGGTTTATTTTCTGGTGGCCTCAT [00141] was chemically synthesized, 32 P labeled at the 5' end with T4 polynucleotide kinase (New England Biolabs), and used as a probe to screen a human bone marrow cDNA library [88].
  • RNAs were run on formaldehyde/agarose gel and transferred onto Hybond-N+ nylon membranes (Amersham). They were hybridized in QuickHyb (Stratagene) and washed according to the manufacturer's instructions.
  • cDNA A mixture of mRNA of several Fy(a-b+) individuals, the BRL Superscript Choice system, and oligo(dT) as a primer was used to prepare cDNA.
  • the cDNA was ligated into ⁇ ZAP Il vector and packaged with Gigapack Gold (Stratagene) extract. About 1.9 x 10 6 unamplified cDNA clones were screened with the 32 P-labeled probe described above.
  • cDNA inserts in pBluescript were isolated by the plasmid-rescue method according to manufacturer's protocol. Both DNA strands were sequenced by using vector primers and by primers designed from the sequenced regions of the transcript [88].
  • a 32 P-labeled 24-mer antisense primer from nt 57-80 of the coding strand was extended on Fy(a-b+) mRNA using a preamplification kit (BRL), and the products were separated on a 6% sequencing gel.
  • DARC protein whether cloned or purified from human erythrocytes is then incubated with HIV-1 by means known in the art.
  • the carrier-bound HIV-1 is exposed to candidate neutralizing antibodies.
  • the unbound candidate neutralizing antibodies are then removed by washing the carrier free of unbound antibodies.
  • a cell lacking CD4 a CD4-negative cell
  • the HIV-1 would be retained on the surface of the cell where it could be tested for binding of antibody and the unbound antibody could be washed off by a variety of standard laboratory methods, such as simple centrifugation, or other methods of separating cells from the surrounding liquid milieu.
  • the level of viral activity and concomitant inhibition are then measured by various means known in the art and described herein.
  • Immune complexes (IC)3 formed by HIV/anti-HIV Abs (HIV-IC) are present in plasma and can bind specific FcRs [91 , 92].
  • Complement activation followed by C3 fragment deposition on the HIV-IC allows efficient binding of HIV to cells such as B lymphocytes, which have receptors for C3 fragments, in particular CD35 and CD21 [93].
  • CD35 also called complement receptor 1 (CR1 ), binds C3b and C3bi and is expressed on erythrocytes as well [72].
  • HIV used in all experiments was of HIV-1 type IMB of the North American isolate, cultured on Hut/4-3 cells, a cell line derived from human lymphocytes.
  • Cell supernatants containing the virions were inactivated by incubation for 1 h at 62°C in the presence of 0.02% formaldehyde to preserve the antigenic properties of the virions.
  • Supernatants were separated from cell debris by low speed centrifugation at 400 x g for 15 min and passed through a 0.45- ⁇ m pore size membrane filter (Minisart filter; Sartorius, Gottingen, Germany).
  • the viral suspension was further concentrated using Centhprep centrifugal filter devices (Mr 10,000 cut-off; Millipore, Bedford, MA), the concentrate was ultracentrifuged twice for 1 h each time at 160,000 x g, and the pellet was resuspended in PBS.
  • HIV-1 was purified by gel filtration on a Sephadex G-25 column (Pharmacia Biotech, Uppsala, Sweden). The HIV-1 was stored at - 80 0 C until use [72].
  • Human erythrocytes were purified from fresh blood of healthy volunteers with blood group O using dextran sedimentation. Briefly, the blood was centrifuged at 680 x g for 7 min, and plasma and buffy coat were removed. Remaining blood cells were mixed with 4% dextran and PBS and left on ice for 40 min. After sedimentation, the supernatant was removed with the upper layer of the erythrocytes, and the rest of the purified RBC were washed five times with RPMI 1640 medium (Life Technologies, Basel, Switzerland), each time removing the upper layer of the cells with the supernatant. The final contamination with leukocytes was reduced to ⁇ 1 leukocyte/10,000 RBC [72].
  • HIV/anti-HIV-IC were formed by incubating ⁇ 1.5 ⁇ g of HIV-1 with 85 ⁇ g of anti-HIV Ig anti-human IgG (Southern Biotechnology Associates, Birmingham, AL) in a total volume of 220 ⁇ l in a shaking water bath for 60 min at 37°C.
  • IC opsonization with complement was achieved by incubation with either fresh NHS (dilution, 1/3) or other complement-deficient sera for 7 min at 37°C.
  • the opsonized HIV-IC were purified by sucrose density gradient ultracentrifugation, as previously described. In short, sucrose gradients consisted of five layers of sucrose solutions 50 to 10% in PBS.
  • Opsonized HIV-IC or HIV were overlaid on the gradients and ultracentrifuged for 2.5 h at 116,000 x g. Fractions of 200 ⁇ l were taken from the bottom of the tube, and the cpm of each fraction were determined. Fractions containing purified opsonized HIV or HIV-IC, located near the bottom of the gradient, were then incubated with erythrocytes [72].
  • opsonized HIV-IC or HIV were incubated with erythrocytes at a ratio of 6000 cpm of the opsonized HIV-IC or HIV/2 x 108 erythrocytes for 10 min at 37°C.
  • the experimental conditions were established to correspond to the situation in vivo. In vivo, the number of viruses are up to 100,000 copies/ml blood, i.e., one virus for 104 erythrocytes.
  • the reaction was stopped by adding ice-cold RPMI 1640 and was centrifuged for 3 min at 170 x g. A fraction corresponding to half the final volume was removed, and the pellet of erythrocytes was resuspended in the remaining half [72].
  • the carrier-bound HIV-1 is exposed to candidate neutralizing antibodies.
  • the unbound candidate neutralizing antibodies are then removed by washing the carrier free of unbound antibodies.
  • a cell lacking CD4 a CD4-negative cell
  • the HIV-1 would be retained on the surface of the cell where it could be tested for binding of antibody and the unbound antibody could be washed off by a variety of standard laboratory methods, such as simple centrifugation, or other methods of separating cells from the surrounding liquid milieu.
  • the washed cells are then co-cultured with CD-4 positive target cells (for example, peripheral blood mononuclear cells or H9 T cell lines) to be subsequently subjected to the neutralizing assay.
  • CD-4 positive target cells for example, peripheral blood mononuclear cells or H9 T cell lines
  • the positive control would consist of the CD4-negative cell- HIV-1 complex that would exhibit the expected level of infectivity of co-cultured CD4- positive cells.
  • erythrocytes are used as CD4-negative carriers, the cells themselves could be examined for the effects of antibody-mediated complement activation in the presence of the bound antibodies by hemolyzing the cells in a standard complement lysis assay.
  • Nonnucleated cells such as erythrocytes, lyse with relative ease when exposed to sensitizing antibody and complement. In the case of erythrocytes, this causes release of hemoglobin, which can easily be measured in the supernatant.
  • Simple, direct and automoation-ready procedures for measuring hemoglobin concentration are readily available, such as the QUANTICHROM TM hemoglobin assay kit manufactured by BioAssay Systems (Harward, CA). This system features a "mix- and-read" procedure that involves the addition of a single working reagent and reading the optical density which is easily automated for high-throughput assay.
  • the method utilizes a Triton/NaOH method whereby the hemoglobin is converted into a uniform colored end product.
  • the intensity of color, measured at 400 nm, is directly proportional to hemoglobin concentration in the sample.
  • measuring the lysis of living nucleated cells is more challenging.
  • Nucleated cells unlike erythrocytes, do not contain a natural chromophore.
  • Nucleated cells have active budding processes to shed membrane attack complexes (MAC) from the membrane and they have ion-pumps that actively reclaim released ions, stabilizing the osmotic balance and preventing rupture of the plasma membrane.
  • MAC membrane attack complexes
  • Virus bound to CD4- cells was up to 17 times more infectious for T cells in cocultures than was the same amount of cell-free virus.
  • the enhanced infection of T cells by virus bound to CD42 cells was not due to stimulatory signals provided by CD42 cells or infection of CD42 cells.
  • anti-CD18 antibody substantially reduced the enhanced virus replication in T cells, suggesting that virus that bound to the surface of CD42 cells is efficiently passed to CD41 T cells during cell-cell adhesion.
  • T-lymphocytic H9 (HTB-176) and B-lymphocytic Raji (CCL-86) cell lines used were obtained from the American Type Culture Collection (ATCC; Manassas, Va.). Cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Whittaker M. A. Bioproducts, Walkersville, Md.) and gentamicin (Sigma, St. Louis, Mo.) at 50 mg/ml.
  • Antibodies to leukocyte function-associated antigen type 1 b (LFA-I b; CD18) and CD14 were obtained from the TS1/18.1.2.11 hybridoma (HB- 203; ATCC) and the 261 C hybridoma (HB-246; ATCC), respectively. Each antibody was purified using an Affinity Pak Immobilized Protein A column (Pierce, Rockford, III.) [74].
  • PBMC obtained from healthy donors were isolated by Ficoll-Hypaque gradient centhfugation (Whittaker M. A. Bioproducts). Stimulated PBMC were produced by culture in medium containing phytohemagglutinin (PHA; 3.0 mg/ml) for 2 days, followed by culture in medium containing interleukin-2 at 20 U/ml. Human recombinant interleukin-2 was obtained through the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health, from Maurice Gately, Hoffman-La Roche, Inc. [94]. Erythrocytes were collected from the bottom layer of the density gradients and washed twice with phosphate- buffered saline.
  • Neutrophils were isolated from healthy donors by Ficoll-Hypaque centhfugation. The bottom layer was collected, and erythrocytes were lysed by three treatments with ice-cold deionized water, followed by treatment with 2x Hanks balanced salt solution containing 5 mM HEPES buffer (Gibco BRL, Grand Island, N.Y.). To isolate platelets, blood was drawn in acid-citrate-phosphatedextrose anticoagulant (Biowhittaker) and centrifuged at 400 x g.
  • the plasma fraction containing platelets was centrifuged (800 x g), washed twice with acidcitrate-phosphate-dextrose in 0.85% NaCI solution (1 :6, vol/vol), and resuspended in 0.85% NaCI until use, when the platelets were resuspended in complete medium [74].
  • CD4+ T cells were positively isolated from fresh PBMC using anti-CD4 antibody-conjugated Dynabeads M-450 and CD4/CD8 DETACHaBEAD (Dynal, Oslo, Norway) in accordance with the manufacturer's protocols. After an additional positive selection of CD4+ T cells, the remaining cells were used as CD4- PBMC. The composition of the CD4+ and CD4- cell population as measured by flow cytometry, was .99% CD4+ and ,1 % CD4+ [74]. Virus stocks.
  • T-cell line-adapted (TCLA) HIV-1 strain MN HIV-1 MN; AIDS Research and Reference Reagent Program [no. 317], contributed by Robert GaIIo
  • the 8E5/LAV cell line AIDS Research and Reference Reagent Program [no. 95], contributed by Thomas Folks
  • the primary isolates of HIV were produced in PHA-stimulated PBMC as previously described [96].
  • a pellet of 5 3 106 erythrocytes or platelets or 1 3 106 Raji cells, neutrophils, total PBMC, CD4-depleted PBMC, purified CD4+ T cells, or tonsil mononuclear cells was incubated with 50 ml of virus containing approximately 1 ,000 pg of p24 for 2 h on ice. Cells were washed to remove unbound virus and transferred to fresh tubes since some virus binding to tubes occurred during incubation.
  • Pelleted cells were treated with 0.5% Triton X-100, and the amount of virus bound to cells was detected by p24 antigen enzyme-linked immunosorbent assay (ELISA; National Institutes of Health AIDS Vaccine Program, Frederick, Md.).
  • ELISA enzyme-linked immunosorbent assay
  • the protease inhibitors leupeptin (2 ⁇ g/ml), aprotinin (10 ⁇ g/ml), and phenylmethylsulfonyl fluoride (2 mM; Sigma) were added to the Triton X-100 solution.
  • Tonsil-derived cells and PBMC were gamma irradiated with 5,000 rads [74].
  • Pelleted Raji cells were first fixed in a 0.5% formaldehyde solution for 10 min at room temperature. Cells were washed four times with serum-free medium, resuspended in complete medium, and then incubated with virus.
  • the HIV-1 virus is allowed to bind to a carrier in such a manner that the carrier-bound HIV-1 retains the ability to infect a target cell. Prior to such an infection the carrier-bound HIV-1 is exposed to candidate neutralizing antibodies. The unbound candidate neutralizing antibodies are then removed by washing the carrier free of unbound antibodies.
  • a cell lacking CD4 (a CD4-negative cell) were to serve as a carrier for binding to HIV-1 , and if the CD4- negative cell could not be infected by the HIV-1 , the HIV-1 would be retained on the surface of the cell where it could be tested for binding of antibody and the unbound antibody could be washed off by a variety of standard laboratory methods, such as simple centhfugation, or other methods of separating cells from the surrounding liquid milieu.
  • Washed cells with bound virus (see above) or dilutions of cell-free virus were added to cultures of 2.5 x 10 5 H9 cells or PHA-stimulated PBMC in round-bottom polypropylene tubes (12 by 75 mm; Becton Dickinson, Franklin Lakes, N.J.). After 12 h, the cells were washed and cultured in 48-well cell culture plates (Corning Inc., Corning, N.Y.) for an additional 6 days and collected on day 7 [74].
  • HIV-1 replication was assessed by measuring the p24 antigen in culture supernatants.
  • anti-LFA-1 ⁇ or anti- CD14 antibody was added at 1 ⁇ g/ml to H9 cells or PHA-stimulated PBMC 20 min prior to coculture with cell-bound virus.
  • Antibody was maintained at 1 ⁇ g/ml throughout the coculture.
  • P24 Antigen Assays as known in the art and described herein, may be applied [74].
  • IMDM Iscove's Modified Dulbecco's Medium
  • PBMC Peripheral Blood Mononuclear Cells
  • PBS Phosphate Buffered Saline
  • TNC Tri Sodium Citrate
  • PHA Phytohaemagglutinine
  • FCS Fetal Calf Serum
  • TCID50 50% Tissue culture infectious dose
  • PBMCs are isolated from buffycoats from 8 - 12 different HIV-negative healthy blood donors. PBMCs from each donor are isolated separately and are simultaneously tested for the presence of the CCR5 32bp deletion. PBMCs from donors homozygous for the 32bp deletion are excluded from the PBMCs pool.
  • erythrocytes When many erythrocytes are present, these can be removed by cell lysis. Resuspend the cells in 1 ml ACK lysing buffer before the last wash step, agitate gently for 1 minute, add up to 50 ml PBS/ 10% TNC, and centrifuge the tubes for 10 min at 250xg (acceleration 9, break 9) at RT.
  • PBMCs of different donors are pooled and frozen at a concentration of 50 million cells/ampoule (1.8 ml) in IMDM supplemented with 20% FCS and 10% DMSO.
  • PBMC serum neutralization assays are performed in triplicate in 96-wells plates, using 20 the carrier-virus complex and 105 PBMCs per well, in IMDM supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 ⁇ g/ml), ciproxine (5 ⁇ g/ml), rlL2 (20 units/ml), and polybrene (5 ⁇ g/ml).
  • Two different protocols are in use, depending on the nature of the neutralizing agent (polyclonal or monoclonal). For polyclonal agents, smaller working volumes are used than for monoclonal agents.
  • a wash step is included in the polyclonal assay to reduce cytotoxic or other unwanted effects of the polyclonal reagent. In both protocols, virus production in culture supernatant at day 7 is analyzed by an in-house ELISA.
  • HIV RG RT PCR (Artus) (24 tests) 10 packs
  • the buffy coat is diluted with equal volume of PBS and then 30 ml of cell suspension is gently placed on the top of 15 ml Ficoll-hypaque in a 50 ml centrifuge tube. Centrifuge at 1 ,500 rpm for 20 min. The PBMCs at interface are collected and transferred to new tube and wash with RPMI 1640. After that, the PBMCs are added with PHA medium for adjust to 1x106 cells/ml. The PBMCs are maintained in tissue culture flask at 37 0 C, 5% CO2. After 2-3 days, the PHA medium is removed and replaced with IL-2 medium for 1 day before adding to HIV-1 co-culture or TCID 50 testing or neutralization test.
  • Carrier complex viral stock is prepared by infection of PHA stimulated PBMCs with 200 ⁇ l of HIV-1 positive supernatant. After overnight incubation, the infected PBMCs are washed with RPMI 1640. Then 4 ml of IL-2 medium are added and cultured in tissue culture flask at 37 0 C, 5% CO2. The p24 antigen is detected by ELISA kit (Organon Teknika) every week. The culture medium is harvested as soon as the p24 positive in high titer. Then TCID 50 is determined and stored the supernatant at -80 0 C for neutralization test.
  • Tissue culture infectious dose (TCID50)
  • TCID50 is calculated according to the Spearman-Karber formula (see Validation of Pharmaceutical Processes By James P. Agalloco, Frederick J. Carleton Edition: 3, illustrated Published by CRC Press, 2007, pp 170-171.)
  • This method estimates neutralization of HIV-1 positive serum or plasma by fixing antibodies in serial dilutions of viral carrier complex. Then the viral load of each dilution is obtained at day 2 by using real time RT-PCR kit (Artus). The ratio of TCID50 of seronegative serum and test serum is calculated and reported.
  • the values of viral load and p24 are collected and analyzed.
  • the neutralizing index is expressed as a ratio of the TCID50 obtained in the presence of
  • the neutralizing index ⁇ 3 is considered negative, while neutralizing index 3-9, 10-100 or >100 are considered as weak, medium and strong neutralizing activity, respectively.
  • Viral load detection by real time RT-PCR 140 ⁇ l of supernatant from neutralization test are extracted by QIAamp viral RNA mini kit (QIAGEN) and RNA is eluted by 60 ⁇ l of elution buffer. This RNA is collected and kept at -80 oC until use. 8 ⁇ l of RNA are used to determine viral load.12 ⁇ l of master mix in HIV 1 RG RT PCR kit (Artus) and 8 ⁇ l of RNA are added in LightCycler capillary tube. Then RT-PCR is performed and using Taqman probe which specific for HIV-1 for detect viral load at channel F1/F2.
  • IL-2 medium Mix 90 ml of RPMI 1640 medium, 10 ml of Fetal bovine serum, 100 ⁇ l of IL-2 (10,000 U/ml), 1 ml of L-glutamine, 200 ⁇ l of penicillin (50,000U/ml) and 200 ⁇ l of Streptomycin (50,000 U/ml).
  • PHA medium Mix 90 ml of RPMI 1640 medium, 10 ml of Fetal bovine serum, 20 ⁇ l of PHA (2.5 mg/ml), 1 ml of L-glutamine, 200 ⁇ l of penicillin (50,000U/ml) and 200 ⁇ l of Streptomycin (50,000 U/ml).
  • EXAMPLE 6 PBMC Neutralization assay using HIV-1 p24 Antigen
  • virus infection in donor PHA- stimulated PBMC is assessed by a quantitative ELISA measurement of HIV-1 p24 antigen expressed in PBMC culture supernatants.
  • Target cells Ficoll gradient separated PBMC from an H IV-1 -seronegative donor.
  • virus carrier complex stock a stock of virus carrier complex stock. Dilute it with IL-2 medium to the concentration calculated from the viral titration. Add 25 ⁇ l of diluted viral stock to the side of each well of the deep-well plate. Be careful not to touch the tips to the plate or the serum at the bottom of the wells. After dispensing virus carrier complex gently tap the deep-well plate to ensure that serum and viral complex are well mixed in the bottom of the wells. Incubate plates at 37°C for thirty minutes.
  • ABL p24 ELISA kit determine p24 concentration in Virus Only row of the experiment plate If the concentration is > 10 ng/ml four days after infection, analyze corresponding sera samples. The harvest p24 plate can be stored at -20 0 C until p24 determination is complete. If the experiment needs to be continued, ⁇ 10 ng/ml p24, add 100ml of fresh IL-2 media to each well of the plate and incubate at 37°C.
  • This assay can be used to detect HIV-1 gp120 proteins or anti-gp120 antibodies.
  • an antibody D7324 adsorbed on a microtiter plate captures gp120 from solution.
  • the captured gp120 protein is then recognized at a separate site by a second antibody that is, in turn, detected using a third, enzyme labeled, anti-antibody.
  • the sandwich ELISA utilized an alkaline-phosphatase (AP) label and the AMPAK ELISA amplification system in the color-development stage1 ,2,3.
  • AP alkaline-phosphatase
  • AMPAK ELISA amplification system in the color-development stage1 ,2,3.
  • Capture antibody Product code D7324 supplied by Aalto Bio Reagents, Dublin, Ireland, is produced by first immunizing sheep with a single synthetic peptide which has the amino acid sequence : APTKAKRRVVQREKR
  • This amino acid sequence corresponds to amino acid numbers 497-511 in the envelope gene gp120 protein of the BH-10 strain of HIV-1.
  • the antibodies are isolated from the sheep hyperimmune serum by affinity chromatography using the above synthetic peptide coupled to Sepharose.
  • any specific anti-gp120 MAb can be used provided that it is able to recognize the particular gp120 protein under evaluation and has a high enough affinity.
  • the bound MAb is then detected using an enzyme-labeled anti-antibody of appropriate specificity. This protocols covers the use of AP- label anti-antibody.
  • the antibody preparation is titrated then the bound antibodies are detected as described above.
  • HIV-1 gp120 Standard [00302] Recombinant HIV-1 gp120 Antigen, product code BR 6106, supplied by Aalto Bio Reagents, Dublin, Ireland.
  • D7324 is reconstituted in distilled water at 1 mg/ml and stored in frozen aliquots.
  • D7324 is coated onto lmmulon Il microelisa plates (Dynatech Ltd.) by incubating them for 12-18 hours at room temperature in 10O ⁇ l per well of 10OmM NaHCO3,.
  • the optimal antibody concentration is 5 ⁇ g /ml.
  • TBS Tris-Buffered Saline
  • 2% non-fat milk powder Marvel, Cadbury Ltd.
  • HIV-carrier complex sample is added to the wells in 10O ⁇ l of TBS (a detergent such as 1 % NP40 can be added but it is usually not necessary) and incubated for 2 hours at room temperature.
  • TBS a detergent such as 1 % NP40 can be added but it is usually not necessary
  • Unbound protein is removed washing twice with TBS (200 ⁇ l) and captured gp120 is detected by addition for 1 hour of a second antibody, as outlined above.
  • the antibody is diluted in TMT/SS buffer (4% nonfat milk powder and 0.5% Tween-20 in TBS plus 20% sheep serum, 10O ⁇ l per well).
  • concentration of detection antibody to be used must be determined empirically, e.g., by titration.
  • the HIV-1 gp120 assay is calibrated using known amounts of purified recombinant HIV-1 gp120 Antigen (product code BR 6106). It is recommended to assign a formal HIV-1 gp120 concentration for product code BR 6106 with reference to a commercial assay for which formally assigned standards are available.
  • the assay is a twin-site sandwich ELISA. Briefly, p24 antigen is captured from a detergent lysate of virions by a polyclonal antibody adsorbed to a solid phase. Bound p24 is detected with an alkaline phosphatase-conjugated anti-p24 monoclonal antibody and a luminescent detection system. The luminescence readout gives a broader dynamic range (2.5 logs versus 1 log with the AMPAK system) and an extended linear range which allows more accurate quantification. Furthermore, the broader dynamic range in this system makes the testing of serial dilutions of samples unnecessary in most cases. It also makes it unnecessary in most cases to re-analyze samples because their p24 content is out of linear range. This reduces the overall number of ELISA samples to be tested and minimizes handling and sample preparation times.
  • Secondary conjugate antibody code BC 1071 -AP alkaline phosphatase conjugate of anti-HIV-1 -p24 mouse monoclonal, 50 ⁇ l/vial; store at +2 to +8 0 C. This is supplied by Aalto Bio Reagents Ltd., Dublin, Ireland.
  • This antibody is an alkaline phosphatase conjugate of monoclonal clone EH12E1.
  • EH12E1 is a mouse monoclonal antibody raised against HIV-1 (CBL-1 ) and mapped to a complex epitope incorporating two distinct peptide sequences as follows :
  • GHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQ (aa 193-227); and, [00320] NPPIPVGEIYKRWII (aa 253-267).
  • TROPIX ELISA-Light Immunoassay system EL100CX chemiluminescent substrate for alkaline phosphatase with enhancer (CSPD with Sapphire-ll)
  • 10 x PBS for 10 liters 80Og. NaCI; 20 g. KCI; 144 g. Na2HPO4; 24 g. KH2PO4; fill up to 8 liters with H20; adjust to pH 7.4 with HCI; fill to 10 liters.
  • p24 Standard Recombinant HIV-1 p24 from Aalto Bio Reagents, Code AG 6054; Dilute with 1 % FCS in TBS to 100 ⁇ g/ml; Store in 100 ⁇ l aliquots at -20 0 C.
  • Sheep serum SIGMA Cat No S-7773
  • Plates COSTAR white opaque 96-well plates, high binding (Cat No 3922) Assay Coating p24 Plates.
  • Day 2 Prepare 500ml 2% milk in 1 x TBS: Stir for one hour at room temperature; Filter twice with Reeve Angel filter paper. [00341] Wash plates twice with 1 x TBS (200ml buffer per well). Add 100ml/well of 2% milk, then stack plates, place a cover plate on top and with saran wrap to avoid evaporation and incubate for one hour at room temp.
  • HIV-1 -Carrier-Neutralizing Antibody Complex Samples to be analyzed should be treated with 1 % Empigen prior to assaying to inactivate the virus and linearize the proteins. (Note: The addition of the detergent is necessary. Omitting the detergent treatment or using other detergents like Tween-20 and NP40 seriously inhibits capture by D7320).
  • Blocking buffer 0.4g milk powder in 20ml IxTBS 2g milk powder in 100ml enough for 4-5 plates.
  • a suitable method of medium-term storage after receipt is to dilute 0.1 ml of the biotinylated conjugate to 1.0ml (1 :10) with TMT / SS buffer. Then aliquot and store 0.1 ml volumes (sufficient for 1 plate) at -20 0 C. Prior to use, dilute 0.1 ml to 10ml (1 :100) in TMT / SS buffer.
  • Coating the plate Add 100 ⁇ l of D7320 (diluted to 1 mg/ml) to 9.9ml coating buffer and add 10O ⁇ l to each well. Cover and leave overnight at room temperature.
  • K Black-bottom, flat-bottom 96-well microplate (Culture Plate 96-F) (Perkin
  • TZM-bl a luciferase expressing reporter cell that possesses CD4 and both CXCR4 and CCR5 coreceptors
  • DMEM DulbeccoA/ogt Modified Eagle's Minimal Essential Medium
  • FBS Fetal Bovine Serum
  • TZM-bl Neutralization Assay [00404] 1. Dilute test neutralizing reagents to desired concentrations.
  • the first microplate for each assay should be a black, clear-bottom view plate. Additional microplates can be the black-bottom culture plate.
  • Britelite reconstitution buffer 1 OmIs buffer into a 10ml substrate bottle. Mix and keep at room temperature.
  • the assay utilized erythrocytes, separated from a leukopack, as virus carrier.
  • the erythrocytes were washed 3 times in PBS, stored in Adsol solution at 4oC until use.
  • Two milliliters of the erythrocyte suspension (in 5x109/ml density) in RPMI medium and 2 ml of the HIV-1 stock (BZ167) were mixed together in a 14 ml polypropylene tube, incubated at 4 0 C for two hours, then washed three times in PBS each followed by a centhfugation at 1500 rpm at 4 0 C.
  • the erythrocyte-bound viruses were resuspended in RPMI medium, and 100 ⁇ l of the suspension was transferred to a polypropylene deep-96-well plate. Twenty microliters of the human monoclonal IgG mAb, b12, was added to each corresponding well in fourfold dilutions (0.06 - 64 ⁇ g/ml). The carrier-virus was incubated with the mAb at 37 0 C for 1 hour, then washed in IL-2 RPMI, and 100 ⁇ l of the erythrocyte-virus-antibody solution was transferred to a polypropylene cell culture plate. Fifty microliters of PBMC as target cell were added to each well.
  • the virus transferred to the target cells, thereafter causing infection in trans.
  • the p24 as a hallmark of the amount of the progeny viruses were measured by the standard p24 capture ELISA kit (ABL Inc.). The manufacturer's protocol was followed with a slight modification. Briefly, viral particles in the culture supernatant were disrupted, inactivating the virus and releasing the p24 into solution to enable detection. The microtiter wells were coated with two murine monoclonal antibodies that react with HIV-1 p24. Test samples were added to the wells, and unbound materials were then thoroughly washed away.
  • the conjugate containing peroxidase-conjugated human anti-p24 polyclonal antibodies were added. After washing away the unbound conjugate, the peroxidase substrate was added. The color intensity were quantified by reading the absorbance at 450 nm. The concentrations of p24 in the samples were interpolated from a p24 standard curve.
  • type 1 uses lipid raft-colocalized CD4 and chemokine receptors for productive entry into
  • HIV-1 coreceptors roles in viral entry, tropism, and disease. Annu Rev Immunol, 1999.
  • Nonneutralizing antibodies are able to inhibit human immunodeficiency virus type 1 replication in macrophages and immature dendritic cells. J Virol, 2006. 80(12): p. 6177-81.
  • GaIiIi U., et al., A unique natural human IgG antibody with anti-alpha-galactosyl specificity. J Exp Med, 1984. 160(5): p. 1519-31.
  • Nichols, M. E., et al. A new human Duffy blood group specificity defined by a murine monoclonal antibody, lmmunogenetics and association with susceptibility to Plasmodium vivax. J Exp Med, 1987. 166(3): p. 776-85.

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Abstract

A unique assay is disclosed for evaluating neutralization of HIV -I virus. The assay uses a carrier cell that contains a lipid bilayer with lipids that allows binding of HIV-I but that cannot be infected by virus. The carrier-bound HIV-I still retains the ability to move from the carrier to a target cell when it is co-cultured with the target cell. In the assay, the carrier-bound HIV-1 is exposed to candidate neutralizing antibodies, and non-bound antibodies are washed away. The carrier-bound HlV-l containing bound antibody is then evaluated for the ability to infect any relevant target cell (such as a CD4-positive lymphocyte), and the degree of infectivity of the target cell will reflect the relative neutralizing activity (if any) of the antibody that was used to coat the carrier-bound HIV-1.

Description

CARRIER NEUTRALIZATION ASSAY
RIGHTS
[0001] This invention was made with support from the United States Government, specifically, the United States Army Medical Research and Materiel Command, and, accordingly, the United States has certain rights in this invention.
CROSS-REFERENCE TO RELATED APPLICATION
[0002] This application claims the benefit of Provisional Application No. 61/070,620, filed March 20; 2008 the contents of which are hereby incorporated by reference.
FIELD AND BACKGROUND
[0003] This invention relates generally to a virus neutralization assay, and specifically to a neutralizing antibody carrier assay wherein said carrier may, by way of nonlimiting example, be a cell with a lipid bilayer operable to bind with HIV-1 but, itself, incapable of infecting a target cell. [0004] Viruses must replicate intracellular^ and often employ host cell enzymes, macromolecules, and organelles for the synthesis of virus particles. Therefore, safe and effective anti-viral compounds must be able to discriminate with a high degree of efficiency between cellular and virus-specific functions. In addition, because of the nature of virus replication, evaluation of the in vitro sensitivity of virus isolates to antiviral compounds must be carried out in a complex culture system consisting of living cells (e.g. tissue culture). The results from such assay systems vary widely according to the type of tissue culture cells which are employed and the conditions of assay. Viral drug resistance is a substantial problem given the high rate of viral replication and mutation frequencies. No disease better exemplifies the problem of viral drug resistance than AIDS.
[0005] Acquired immune deficiency syndrome (AIDS) is a fatal human disease, generally considered to be one of the more serious diseases to ever affect humankind. Globally, the numbers of human immunodeficiency virus (HIV) infected individuals and of AIDS cases increase relentlessly and efforts to curb the course of the pandemic, some believe, are of limited effectiveness. Historically, vaccines that induce antibodies have been the most effective strategy to combat viral diseases such as polio, hepatitis, measles, and influenza. While antibodies are known to play an important role in protection in these diseases, the effectiveness of antibodies in human immunodeficiency virus type 1 (HIV-1 ) protection has yet to materialize.
HIV Structure and Genome [0006] HIV-1 is a spherical, enveloped RNA retrovirus, a lentivirus, that fuses with the plasma membrane of a host cell to insert its genomic RNA. The envelope of HIV-I contains a lipid bilayer that is associated with two loosely bound glycoproteins, gp120 and gp41. These proteins are created during intracellular virus assembly when a precursor protein, gp160, is cleaved to from pg120 and gp41 (see FIG. 1A). The gp41 is a trimeric transmembrane protein that is anchored in the lipid bilayer, and during viral maturation and budding the intraviral end of gp41 is bound to sites located on an N- terminal myristoylated matrix protein (p17) [1 -3].
[0007] HIV-1 has a diploid genome having two identical RNA molecules. The molecular organization of HIV is (51) U3-R-U5-gag-pol-env-U3-R-U5 (31). The U3, R, and U5 sequences form the long terminal repeats (LTR) which are the regulatory elements that promote the expression of the viral genes and sometimes nearby cellular genes in infected hosts. The internal regions of the viral RNA code for the structural proteins: gag (p55, p17, p24 and p7 core proteins), pol (p10 protease, p66 and p51 reverse transcriptase and p32 integrase) and env (gp120 and gp4l envelope glycoproteins). Gag codes for a polyprotein precursor that is cleaved by a viral protease into three or four structural proteins; pol codes for reverse transcriptase (RT) and the viral protease and integrase; env codes for the transmembrane and outer glycoprotein of the virus. The gag and pol genes are expressed as a genomic RNA while the env gene is expressed as a spliced subgenomic RNA. In addition to the env gene there are other HIV genes produced by spliced subgenomic RNAs that contribute to the replication and biologic activities of the virus. These genes include: tat which encodes a protein that activates the expression of viral and some cellular genes; rev which encodes a protein that promotes the expression of unspliced or single-spliced viral mRNAs; nef which encodes a myhstylated protein that appears to modulate viral production under certain conditions; vif which encodes a protein that affects the ability of virus particles to infect target cells but does not appear to affect viral expression or transmission by cell-to-cell contact; vpr which encodes a virion-associated protein; and vpu which encodes a protein that appears to promote the extracellular release of viral particles.
HIV Replication Mechanics
[0008] The human immunodeficency virus type 1 (HIV-1 ) has a lipid bilayer that contains several glycoproteins that are anchored in, or closely associated with, the membrane surface. The envelope proteins have complex interactions with the lipids both on the host cells and on the target cells. The processes of budding from host cells and entry into target cells occur at sites on the plasma membrane, known as lipid rafts, that represent specialized regions that are rich in cholesterol and sphingolipids. As with other enveloped viruses, the lipid_composition of the virus is largely reflective of the composition of lipid rafts present in the plasma membrane of the host cell [4, 5]. Structure, function, and lipid composition of HIV-1 and lipid interactions of HIV-1 with cells [6]. [0009] It is now widely accepted that HIV-I buds out of and enters into the lipid raft areas of the plasma membrane (see FIG. 1 ) [7-12]. HIV-1 also uses lipid raft- colocalized CD4 and chemokine receptors for productive entry into CD4+ cells [13]. Lipid raft glycosphingolipids that have relevance to HIV -1 budding or entry include, among others, gangliosides GM 1 and GM3, GalCer, SGalCer, LacCer, and CTH (see FIG. 2).
[0010] After budding from host cells, the HIV-1 virus exhibits a strong tendency to infect T lymphocytes as target cells, using CD4 as a receptor [14]. The binding and fusion of HIV with the target cell involves a choreographed ballet between the proteins of the free virus and the entry site of the target cell (FIG. 1 B). HIV entry into a cell is a multistep process initially involving the interactions of viral envelope protein gp120 and gp41 with several binding sites on the cell surface. The envelope proteins exist as a trimer consisting of gp120 molecules and 3 gp41 molecules. The binding of gp120 to CD4 is followed by conformational changes in the gp120 protein that expose binding sites to chemokine receptors, CXCR4 or CCR5, that serve as co-receptor binding sites for interactions of the virus with the target cell [15-17]. The binding of gp120 to the chemokine co-receptor, in turn, induces conformational changes that allow the binding of the gp41 anchor protein to the cell, and this is followed by fusion of the viral lipid bilayer with the plasma membrane bilayer, and entry of the virion RNA into the target cell [18] (see FIG. 1 C). The binding and entry processes entail numerous types interactions between proteins and lipids of the virus and specific lipids of the target cell [19]. Roles of Glycosphingolipids In HIV-1 Interactions With Host Cells
[0011] Several glycosphingolipids, including GalCer and SGalCer have been proposed as alternate receptors or binding sites for HIV-1 [20-25], using a truncated form of gp120, proposed that the binding site for GalCer/SGalCer is located between amino acids 206-275. In contrast, a binding site of HIV-1 to GalCer has also been located to residues 650-685 of gp41 , and more specifically to residues 662-667 (comprising ELDKWA) on gp41 which is proposed to serve as a lectin for galactose-specific binding of HIV-1 to epithelial cells [20]. GM3 and CTH have also been indicated as binding sites for HIV-1 envelope [26-28]. Interestingly, the V3 loop on gp120 has been reported to have a binding site for GalCer [29], and gp120 also binds GM3 and CTH [26-28]. This therefore indicates that gp120 and gp41 might both bind to GalCer, SGalCer, GM3, and CTH. It is conceivable that this could represent a sequential interaction of gp120 with the lipid bilayer, followed by interaction of gp41 with the lipid bilayer during a fusion process. This is suggested by the binding of GM3 to both the V3 loop and to CD4 [27]. The GM3 binding to V3 involves the sequence GPGRAF, while GM3 binds CD4 through a different sequence [27]. Thus, it has been proposed that GM3 facilitates the interaction of the V3 loop with CD4 [27]. It should also be noted that the precise pg120 binding site for every glycolipid has not necessarily been delineated, and it is possible that sites other than GPGRAF might be involved in the binding of certain glycolipid headgroups. Vaccine Strategies for Developing Antibodies That Would Be Effective For Blocking HIV-1
[0012] One of the major barriers that has emerged in the development of an effective HIV-1 vaccine is the difficulty in obtaining neutralizing antibodies that block infection by field isolates derived from a wide cross-section of clades (subtypes) [30, 31]. Although the envelope glycoproteins are antigenic molecules that potentially might be used for development of broadly neutralizing antibodies in a vaccine to HIV-1 , the development of such antibodies that have broad specificities against primary field isolates of virus have been largely thwarted to date by the ability of the envelope proteins to evade the immune system through various mechanisms.
Assays for Detecting Neutralizing Antibodies to HIV-1
[0013] Evidence in favor of a beneficial effect of HIV-1 neutralizing antibodies has been presented over the years [32-37]. Despite this, early moves towards vaccine clinical studies in the early 1990s were discouraged by the limited titer and very narrow specificity of neutralizing antibodies induced by natural infection or immunization if neutralization was detected at all [38]. Furthermore, the high level of genetic variability of the virus and its escape from the neutralizing antibody response are well documented and have further discouraged the HIV-1 vaccine field from considering the induction of humoral immunity as a pre-requisite for an effective HIV-1 vaccine [39]. Consequently, in the late 1990s and the early years of this century vaccine efforts were mainly focused on eliciting a cellular immune response but, unfortunately, these have also failed to provide effective protection against HIV-1 [40, 41].
[0014] As a result, AIDS vaccine development has developed a renewed emphasis on the potential role for neutralizing antibodies in a successful global vaccine. As a result, development of a standardized platform for reproducible measurement of neutralizing antibodies has received considerable attention.
[0015] Most effective viral vaccines work, at least in part, by generating antibodies that inactivate or neutralize the invading virus, and the existing data strongly suggest that an optimally effective HIV-1 vaccine should elicit potent antiviral neutralizing antibodies. However, unlike acute viral pathogens, HIV-1 chronically replicates in the host and evades the antibody response. This immune evasion, along with the large genetic variation among HIV-1 strains worldwide, has posed major obstacles to vaccine development. Current HIV vaccine candidates do not elicit neutralizing antibodies against most circulating virus strains, and thus the induction of a protective antibody response remains a major priority for HIV-1 vaccine development. For an antibody- based HIV-1 vaccine, progress in vaccine design is generally gauged by in vitro assays that measure the ability of vaccine-induced antibodies to neutralize a broad spectrum of viral isolates representing the major genetic subtypes (clades) of HIV-1 [42]. Although the virus envelope (Env) proteins have evolved an extraordinary ability to evade neutralizing antibodies, a vaccine that can elicit protective antibodies remains the best hope for developing an HIV vaccine that confers sterilizing immunity [43]. [0016] The main targets for neutralizing antibodies to HIV-1 are the surface gp120 and trans-membrane gp41 envelope glycoproteins (Env) that mediate receptor and coreceptor binding and the subsequent membrane fusion events that allow the virus to gain entry into cells [44]. Antibodies neutralize the virus by binding these viral spikes and blocking virus entry into susceptible cells, such as CD4+ T cells [45, 46]. In order to chronically replicate in the host, the virus exploits several mechanisms to shield itself against antibody recognition, including a dense outer coating of sugar molecules (N- linked glycans) and the strategic positioning of cysteine-cysteine loop structures on the gp120 molecule [47].
[0017] Induction of an effective neutralizing antibody response will require that a vaccine deliver to the naϊve B cell repertoire epitopes that are both immunogenic (i.e., possess favorable properties for B cell inductive pathways) and antigenic (i.e., available for high affinity antibody binding on functional Env spikes). Viral epitopes that are conserved among most viral strains are more likely to generate cross-reactive antibodies. In this regard, researchers have focused on a small number of human MAbs, from clade B HIV-1 -infected individuals, that possess broadly cross-reactive neutralizing activity [48]. The cognate viral epitopes for these MAbs have been well characterized and are being evaluated as vaccine immunogens.
[0018] The phenomenon of virus neutralization is a function of three variables: the antibody (Ab), the virus and the target cell. Variation in any one of these parameters may drastically affect the results of assays for neutralization [49]. [0019] Antibody (Ab) mediated neutralization of virus particles renders virions noninfectious for permissive cells. This process of neutralization can be indirect or direct. Indirect neutralization requires secondary factors such as complement to lyse the virion, or cells which interact with the Ab-virus complex, leading, in some cases, to destruction of the virion. Direct neutralization, on the other hand, does not destroy the virus but leads to a loss of infectivity [50]. Abs may aggregate virus particles, rendering them non-infectious, or, with some Abs at saturating conditions, neutralization may occur by preventing virion binding to cells. But many polyclonal and monoclonal antibodies (mAbs) that neutralize do so at a post-binding step. The critical role of Ab specificity is perhaps best illustrated by the fact that (a) an Ab that does not bind to a virus cannot neutralize it, and (b) not all Abs which bind to virions neutralize them.
[0020] In addition to the key roles played by the Ab and the virus in neutralization, the nature of the target cell also is critical to the outcome of the neutralization assay. Thus, each of these components must be defined and analyzed in order to understand the mechanism(s) underlying neutralization and to design neutralization assays with maximum sensitivity.
[0021] Variation in the virus can, predictably, cause profound changes in the neutralization assay. Both the integrity of the virus preparation [51] as well as the nature of the cells used to produce the virus [52] affect the outcome of neutralization. [0022] In order to adequately monitor neutralization breadth and potency and to compare and prioritize immunogens, assays are needed that are sensitive, quantitative, high throughput, and have correlative value. The challenge is to develop standardized in vitro assays that will allow for a meaningful comparison of the quality and potency of neutralizing antibodies in sera or other fluids from HIV-positive patients and vaccine recipients. Importantly, the neutralization assay outcome may be determined by numerous assay parameters. These variable parameters include: target cell used and cell density, host cell used for viral stock propagation, virus dose and antibody dilution/concentration (virus particle: antibody ratio), the inclusion of complement, volumes of components added, duration of pre-incubation of virus and antibody, duration of infection with or without antibody, cell washing steps to remove unbound antibody and virus, length of culture time, the endpoint measured and other variables.
[0023] Neutralization platforms have been evolving over two decades. In the majority of neutralization assays, virus and antibody are incubated together and then added to CD4+ target cells. The critical concern regarding the use of any cell line-based model system is its physiologic relevance and value as a surrogate for in vivo outcomes. Subsequently, assays were developed to infect HIV seronegative peripheral blood mononuclear cells (PBMC) from healthy human donors to address these concerns. However PBMC from different donors display differential susceptibility to HIV-1 infection. These differences in HIV replication may be due, in part, to host genetics and CD8+ cell factors, the number of CD4+ cells or expression levels of CD4 molecules on host cells, effects of host cell-derived molecules on the viral surface, and host genetic polymorphisms in chemokines or chemokine receptors, such as CCR5, which function as HIV-1 coreceptors. Because of these variables, the inter-lab, and even the inter- experiment variation within a single lab, is often quite problematic in PBMC-based neutralization assays.
[0024] A newer approach is to use "pseudoviruses" that incorporate molecularly
cloned HIV-1 Envs into defective virus particles capable of only a single round of infection. The pseudoviruses are generated in the 293T cell line by cotransfection of an env-mutated viral backbone, along with the env clone of choice, and then used to infect a transformed cell line expressing the appropriate receptors. One such format is an assay that employs an epithelial HeLa-dehved cell line (TZM-bl) that carries the luciferase reporter gene sensitive to the presence of the HIV Tat protein. This TZM-bl assay has been advanced as a readily transferable method for assessing vaccine- elicited neutralizing antibodies in the good clinical laboratory practices (GCLP) environment. Table A shows a comparison of several of the variable parameters (as described above) that distinguish the PBMC assay from the pseudovirus approach [53].
TABLE A
Virus Target Cells Measure of Infection
T-cell line adapted Neoplastic CD4+ Syncytia or plaques
T-cell line Cell-killing
Expressing CXCR4 Gag antigen expression Primary isolates Primary human T-CeIIs Gag antigen expression
RT activity
Primary isolates, Genetically engineered Luciferase
Env pseudoviruses, Cell lines expressing Green fluorescent protein
Chimeric infectious CD4, CCR5 and CXCR4 Secreted alkaline phoshatase
Molecular clones β-galactosidase
[0025] The pseudoviral system has several advantages as a platform for application to front-line assessment of antibodies, to include: the ability to rapidly test for neutralization against primary patient Envs from multiple clades, a high degree of inter- experiment reproducibility and throughput, ease and safety of reagent distribution for the assay, and facilitation of assay validation and global transfer. However, specific
discrepancies in the data obtained when reporter cell line-based pseudovirus assays are compared with PBMC-based assays, have recently been.
[0026] Assays employing primary cells typically capture all stages of the virus life cycle during neutralization assays incorporating multiple rounds of infection. The TZM-
bl assay is a single round assay and primarily assesses inhibition of virus binding and entry. It is possible that some antibody subpopulations may not be detected through the use of a single assay focusing only on virus entry. Another fundamental difference between T cells and the HeLa-derived TZM-bl cells may lie in the location of HIV/receptor interaction(s). The majority of HIV-1 entry into T cells occurs at the plasma membrane via CD4 and chemokine receptor-mediated engagement and fusion. In HeLa cells, endocytosis has been reported to account for approximately 85% of virus entry [54]. Moreover, TZM-bl assays often include the use of DEAE-dextran during the infection phase. If endocytosis is playing a significant role in viral entry in TZM-bl cells, this compound most likely enhances productive infection by buffering endosomes, thus allowing the pseudovirus to avoid lysosomal degradation and to enter the cell through
the endosomal vesicle.
[0027] Data suggest that certain cell line models, ie. TZM-bl cells, express vastly different levels of the cell surface coreceptor CCR5, compared to primary PBMC (Table
B) [43].
TABLE B
Assay PBMC TZM-bl (JC53-bl)
Cells PBMC Epithelial HeLa Virus Uncloned primary Pseudovirus
Assay length 4-6 days 2-3 days Common endpoint Extra/lntra-cellular p24 Luciferase activity Rounds of infection Multiple/Single Singly only DEAE-dextran used No Yes Measure of inhibition: Attachment/Entry Yes Yes Cell2cell transmission Potentially No Coreceptors used CCR5, CXCR4 CCR5, CXCR4 (>2 logs more
CCR5 than PBMC [0028] Recent technological advances have provided the means to standardize several aspects of virus neutralization assays. Engineered cell lines expressing high levels of CD4, CCR5, and CXCR4 can be substituted for primary human T cells [55], thus alleviating the requirement for individual donor cells. In addition, it is possible to utilize Env-pseudotyped lentiviral vectors expressing any HIV-1 Env that can be cloned into an appropriate DNA expression plasmid [56, 57]. The Env is expressed in trans with an env-deficient HIV-1 , and the resulting Env-pseudotyped virions produce a single round of infection that can be monitored by reporter genes carried in the virus or the engineered cell line. Cloning of functional env genes from plasma viral RNA or from proviral DNA from primary peripheral blood mononuclear cells (PBMC) or cultured PBMC is possible. The use of appropriate positive and negative control reagents and a rigorous program of proficiency testing can then ensure that assays performed in different laboratories generate equivalent data [53].
Summary of the Problem of Neutralization Assays
[0029] Thus, to summarize, one of the major barriers facing the HIV-1 vaccine field lies in the technical and theoretical difficulties of measurement of neutralizing antibodies. Although numerous proposed neutralizing antibody assays have been created, to date there is no consensus regarding which assay, if any, represents relevant neutralization of virus that could be correlated with protective immunity to field isolates of HIV-I that might be induced by a vaccine. The majority of neutralizing assays utilize cell culture systems in which free virus is co-incubated with target cells. The assays generally consist in the attempted blocking of HIV-1 infection of target cells by incubation of the antibody in the cell culture in such a manner that cell- free virus would be exposed to the antibody. It is widely believed that in order to obtain broadly neutralizing or protective antibodies to HIV-1 it will be necessary for antibodies to utilize antigenic epitopes (i.e., molecular recognition sites for binding of antibodies) that are conserved in the virus or that are present in the host or target cell in the regions in which the virus either buds or where binding or fusion with the virus occurs [31 , 58]. However, in the assays that have been developed it is not known whether the antibodies are reacting directly with the free virus; or with the envelope antigens as they undergo conformational changes during the entry process into target cells; or with the target cells themselves; or with the budding virus as it emerges from the host cell; or with the host cell itself; or even whether the antibody actually enters the target or host cell (e.g., through endocytosis or pinocytosis) where it might interact with the intracellular virus that is maturing within the cell. What is needed is an assay that allows direct interaction of potentially neutralizing antibodies with HIV-1 itself in the absence of host cell or target cell interactions, but which allows subsequent assay of the ability of the HIV-1 that has been exposed to antibody, for the ability (or inability) to infect a target cell. The present invention provides such an assay.
BRIEF DESCRIPTION OF THE PREFERED EMBODIMENT
[0030] A method of performing an assay comprising the steps of: incubating a carrier with a said test sample to produce a first complex; incubating an analyte with said first complex to produce a second complex ; and, performing a measurement on said complex.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In the drawings:
[0032] FIG. 1 is a diagram of the interactions of HIV-1 envelope proteins with plasma membrane lipids during target cell binding (B) and fusion steps (C);
[0033] FIG. 2 is a structural drawing of ceramide and selected gylcosphingolipidsthat bind and interact with HIV-1 envelope proteins, or with CD4 or chemokine receptors, during binding and fusion of HIV-1 with target cells.
[0034] FIG. 3 is a table showing key details of various neutralization assay protocols.
[0035] FIG. 4 is a table showing key differences between neutralization assay protocols.
[0036] FIG. 5 is a diagram of the plasma membrane glycosphingolipid microdomains as preferential sites of formation of the HIV-1 fusion complex;
[0037] FIG. 6 is a schematic diagram of HIV-1 gp41 envelope protein; [0038] FIG. 7 is a model of the HIV-1 molecule showing gp41 at the vicinity of the lipid bilayer.
[0039] FIG 8 is line graph showing percent neutralization of the carrier-neutralization assay as a function of antibody saturation.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0040] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the art.
[0041] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
[0042] It must be noted that, as used in this specification and the appended claims, the singular forms "a" , "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to an antigen includes a mixture of two or more antigens, and the like.
[0043] The following amino acid abbreviations are used throughout the text: [0044] Alanine: Ala (A) Arginine: Arg I Asparagine: Asn (N) Aspartic acid: Asp (D) Cysteine: Cys (C) Glutamine: GIn (Q) Glutamic acid; GIu (E) Glycine; GIy (G) Histidine: His (H) Isoleucine: lie (I) Leucine: Leu (L) Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro (P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr (Y) Valine: VaI (V).
Definitions
[0045] In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below unless otherwise noted:
[0046] "Adjuvant": An adjuvant is defined as anything that will amplify the immune response or improve the immune response over what the immune response would be without the adjuvant.
[0047] "Analyte," as used herein, is the substance to be detected which may be present in the test sample. The analyte can be any substance for which there exists a naturally occurring specific binding member (such as, an antibody), or for which a specific binding member can be prepared. Thus, an analyte is a substance that can bind to one or more specific binding members in an assay. "Analyte" also includes any antigenic substances, haptens, antibodies, and combinations thereof. As a member of a specific binding pair, the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of intrinsic factor protein as a member of a specific binding pair for the determination of Vitamin B12, the use of folate-binding protein to determine folic acid, or the use of a lectin as a member of a specific binding pair for the determination of a carbohydrate. The analyte can include a protein, a peptide, an amino acid, a nucleotide target, and the like.
[0048] An "antigenic epitope" (or, more simply, an epitope): is a molecular recognition site for binding of antibodies. Commonly this is determined or produced by injecting an antigenic material into a mammal, or by introduction of the antigenic material to lymphocytes in vitro, for presentation of the antigenic material to lymphocytes to induce antibodies that are secreted by lymphocytes, and said antibodies then have the capacity to bind to sites on the material that had been presented to the lymphocytes.
[0049] "Broadly neutralizing": A commonly encountered problem in HIV-1 immunology and vaccinology is the inability of antibodies induced against HIV-1 organisms produced in the laboratory to prevent (i.e., neutralize) primary isolates of HIV-1 viruses from infecting target cells. Broadly neutralizing antibodies are defined as antibodies that have the ability to partially or completely overcome this problem by neutralizing more than one type of primary isolate of HIV-1 virus.
[0050] A "Carrier" is a nontoxic, cell, cellular component or molecule to which a virus, pseudovirus and/or viral subunits are conjugated by physiological or biochemical means. [0051] By "Carrier bound HIV-1 ", a carrier as defined herein bound to HIV-1 by physiological or biochemical means.
[0052] A "Candidate Neutralizing Anitbody" means broadly neutralizing HIV antibodies such as, by way of nonlimiting example, 4E10, 2F5, and Z13: These are designations of monoclonal antibodies, derived from individual humans infected with HIV-1 , or that have been identified from phage display libraries, that have the ability to broadly neutralize clinical isolates of HIV-1. The antibodies are further taught and described by Buchacher et al. [Buchacher A, Predi R, Strutzenberger K, Steinfellner W, Trkola A, Purtscher M, Gruber G, Tauer C, Steindl F, Jungbauer A, Katinger H. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Research and Human Retroviruses. April 1994; 10(4):359-369] and by Zwick et al. [Zwick M B, Labrijn A F, Wang M, Spenlehauer C, Saphire E O, Binley J M, Moore J P, Stiegler G, Katinger H, Burton D R, Parren P W, Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. Journal of Virology November 2001 ; 75(22): 10892-10905].
[0053] Dual-specific or multi-specific: This is defined as the ability of an antibody to bind simultaneously or independently to epitopes on two or more types of antigenic chemical species, for example to an amino acid sequence and to a lipid; or to a sugar and a lipid; or to an amino acid sequence and a sugar. The term "dual" refers only to binding to more than one type of chemical epitope, but such antibody binding specificities may actually contain as many molecular binding sites for different types of chemical epitopes (including three, or more, epitopes) as there is available space on the binding site of the antibody for such simultaneous binding of more than one type of epitope.
[0054] "Enveloped virus": A virus that has an envelope (i.e., an outer lipid bilayer structure together with associated proteins on the outer surface) is an enveloped virus. Examples of such viruses include: HIV-1 , influenza virus, dengue virus, Sindbis virus, and Ebola virus, among many others.
[0055] "Enzyme-Linked Immunosorbent Assay" (ELISA), is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shown upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.
[0056] Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates enabling much higher sensitivity.
[0057] The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity. In an ELISA test, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens have been attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to other antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number. [0058] The "Indirect" ELISA. The steps of the general, "indirect," ELISA for determining r antibodies, serum antibody concentrations are:
[0059] Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.
[0060] A concentrated solution of non-interacting protein, such as bovine serum albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.
[0061] The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
[0062] The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins. [0063] Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate- specific enzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
[0064] Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
[0065] Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
[0066] View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device.
[0067] The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is nonspecific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich ELISA provides a solution to this problem. [0068] ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.
[0069] Sandwich ELISA. A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:
[0070] Prepare a surface to which a known quantity of capture antibody is bound.
[0071] Block any non specific binding sites on the surface.
[0072] Apply the antigen-containing sample to the plate.
[0073] Wash the plate, so that unbound antigen is removed.
[0074] Apply primary antibodies that bind specifically to the antigen.
[0075] Apply enzyme-linked secondary antibodies which are specific to the primary antibodies.
[0076] Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
[0077] Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal.
[0078] Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen. [0079] The image to the right includes the use of a secondary antibody conjugated to an enzyme, though technically this is not necessary if the primary antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. The major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.
[0080] Competitive ELISA. A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:
[0081] Unlabeled antibody is incubated in the presence of its antigen.
[0082] These bound antibody/antigen complexes are then added to an antigen coated well.
[0083] The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
[0084] The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme. [0085] A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
[0086] For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal. (Note that some competitive ELISA kits include enzyme- linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal).
[0087] ELISA Reverse method & device (ELISA-R m&d). A newer technique uses a solid phase made up of an immunosorbent polystyrene rod with 4-12 protruding ogives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents. The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and different antigens for multi-target assays; The sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples; [0088] One ogive is left unsensitized to measure the non-specific reactions of the sample; The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating ready-to-use lab-kits and on-site kits. [0089] "Flow cytometric procedures" and particle counting procedures include processes whereby analytes which are antibody members of specific binding pairs are quantified by mixing an aliquot of test sample suspected of containing a specific antibody with microparticles coated with a capture reagent specific for such antibody such as at least one of the peptides disclosed herein, capable of binding to the antibody of interest as the other member of the specific binding pair. If the antibody is present in the test sample, it will bind to some of the microparticles coated with the capture reagent and agglutinates will form. The analyte concentration is inversely proportional to the unagglutinated particle count. See, for example, Rose et al., eds., Manual of Clinical Laboratory Immunology, 3rd edition, Chapter 8, pages 43-48, American Society for Microbiology, Washington, D.C. (1986).
[0090] Flow cytometry methods that sense electronic and optical signals from cells or particles which are illuminated allows determination of cell surface characteristics, volume and cell size. Antibody present in, for example, a test sample are bound to a peptide disclosed herein and detected with a fluorescent dye which is either directly conjugated to the peptide or added via a second reaction. Different dyes, which may be excitable at different wavelengths, can be used with more than one peptide specific to different analytes such that more than one analyte can be detected from one sample. In fluorescence flow cytometry, a suspension of particles, typically cells in a test sample, is transported through a flowcell where the individual particles in the sample are illuminated with one or more focused light beams. One or more detectors detect the interaction between the light beam(s) and the labeled particles flowing through the flowcell. Comnnonly, some of the detectors are designed to measure fluorescence emissions, while other detectors measure scatter intensity or pulse duration. Thus, each particle that passes through the flowcell can be mapped into a feature space whose axes are the emission colors, light intensities, or other properties, i.e., scatter, measured by the detectors. In one situation, the different particles in the sample map into distinct and non-overlapping regions of the feature space, allowing each particle to be analyzed based on its mapping in the feature space. To prepare a test sample for flow cytometry analysis, the operator manually pipettes a volume of test sample from the sample tube into an analysis tube. A volume of the desired fluorochrome labeled peptide is added. The sample/peptide mixture then is incubated for a time and under conditions sufficient to allow antibody/peptide bindings to take place. After incubation, and if necessary, the operator adds a volume of RNS lyse to destroy any RBCs in the sample. After lysis, the sample is centhfuged and washed to remove any left-over debris from the lysing step. The centrifuge/wash step may be repeated several times. The sample is resuspended in a volume of a fixative and the sample then passes through the fluorescence flow cytometry instrument.
[0091] A "hapten" is a small molecule which can elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one which also does not elicit an immune response by itself. The term "hapten", as used herein, refers to a partial antigen or non-protein binding member which is capable of binding to an antibody, but which is not capable of eliciting antibody formation unless coupled to a carrier protein. Haptens may be used to enhance the signal generated, and thus the sensitivity of the assay. The use of haptens is known in the art. It is contemplated that haptens also can be used in assays employing the peptides disclosed herein in order to enhance performance of the assay.
[0092] The "indicator reagent" comprises a "signal generating compound" (label) which is capable of generating and generates a measurable signal detectable by external means conjugated (attached) to a specific binding member for HIV. "Specific binding member" as used herein means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. In addition to being an antibody member of a specific binding pair for HIV, the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like. An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to HIV as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
[0093] "Lipid": Lipids are defined as taught by Small, D. M., "The Physical Chemistry of Lipids, From Alkanes to Phospholipids" Handbook of Lipid Research, VoI, 4, Plenum, NY, 1986, p. 1 , as given below:
[0094] "1.1 Definition of lipids: Assuming a broad definition, one can define a lipid as any molecule of intermediate molecular weight (between 100 and 5000) that contains a substantial portion of aliphatic or aromatic hydrocarbon. Included are the hydrocarbons, steroids, soaps, detergents, and more complex molecules, such as triacylglycerols, phospholipids, gangliosides, and lipopolysaccharides. Immediately, one can imagine that the physical behavior of such chemically divergent molecules will be quite different. Indeed one of the most interesting characteristics of lipids is their tremendously varied behavior in aqueous systems, ranging from almost total insolubility (e.g., paraffin oil and sterol esters) to nearly complete solubility (e.g., soaps, detergents, bile salts, and gangliosides). This particular aspect of lipids is important biologically because all cells exist in an aqueous milieu."
[0095] "Lipid structure" (this includes all organized lipid structures, or domains, and all solid phase, mesomorphic, crystalline, liquid crystalline, and liquid lipid structures): This is defined as all of the multiple organized physical states of lipids, as taught by Small, D. M., in "The physical states of lipids: solids, mesomorphic states, and liquids" in "The Physical Chemistry of Lipids, From Alkanes to Phospholipids" Handbook of Lipid Research, VoI, 4, Plenum, NY, 1986, Chapter 3, pp. 43-87. All of the above terms are interchangeable as defined in the context of this invention. Thus, the term "solid phase lipid structure" is interchangeable with "mesomorphic states", "liquid lipids", "organized lipid structures" "domains", "crystalline lipid structures", liquid crystal lipid structures", and "liquid lipid structures".
[0096] "Lipid bilayer membrane": This is a type of double layer membrane in which the polar groups of the parallel array of lipids of each monolayer of lipids are oriented toward the aqueous phase and the nonpolar groups (such as fatty acyl groups) of each monolayer are oriented toward each other in the center of the bilayer. Liposomes often contain lipid bilayers, as do plasma membranes of cells.
[0097] "Liposomes": Liposomes, as they are ordinarily used, consist of smectic mesophases, and may consist or either phospholipid or nonphospholipid smectic mesophases.
[0098] A "neutralization assays " is an assay format designed to utilize the carrier complexes described herein which contain antigenic epitopes that are useful in competitive assays such as described herein. As general, nonlimiting example of a neutralization assay, a peptide representing an epitope of an antigenic region of HIV-1 , is solubilized and mixed with a sample diluent to a final concentration of between 0.5 to 50.0 .μg/ml. A known amount of test sample (for example, 10 μl), either diluted or non- diluted, is added to a reaction well, followed by, for example, 400 .μl of the sample diluent containing a peptide (such as the carrier complex described herein). If desired, the mixture may be preincubated for approximately 15 minutes to two hours. A solid phase coated with the peptide is then is added to the reaction well, and incubated for one hour at approximately 40 degrees C. After washing, a known amount of an indicator reagent, for example, 200 .μl of a peroxidase labelled goat anti-human IgG in a conjugate diluent, is added and incubated for about one hour at 40 degrees C. After washing and when using an enzyme conjugate such as described, an enzyme substrate, for example, OPD substrate, is added to the mixture and incubated at room temperature for thirty minutes. The reaction is terminated by adding a stopping reagent such as 1 N sulfuric acid to the reaction well. Absorbance is read at 492 nm. Test samples which contain antibody to the specific peptide generate a reduced signal caused by the competitive binding of the peptides to these antibodies in solution. The percentage of competitive binding may be calculated by comparing absorbance value of the sample in the presence of peptide to the absorbance value of the sample assayed in the absence of a peptide at the same dilution. Thus, the difference in the signals generated between the sample in the presence of peptide and the test sample in the absence of peptide is the measurement used to determine the presence or absence of antibody.
[0099] The present day neutralization assay protocols are described above are listed in FIG. 3 and differences between assay protocols are shown in FIG. 4.
[00100] Opsonization" is the process by which a pathogen is marked for ingestion and destruction by a phagocyte. Opsonization involves the binding of an opsonin, i.e., antibody, to a receptor on the pathogen's cell membrane. After opsonin binds to the membrane, phagocytes are attracted to the pathogen. The Fab portion of the antibody binds to the antigen, whereas the Fc portion of the antibody binds to an Fc receptor on the phagocyte, facilitating phagocytosis. The receptor-opsin complex can also create byproducts like C3b andC4b which are important components of the complement system. These components are deposited on the cell surface of the pathogen and aid in its destruction. [00101] The cell can also be destroyed by a process called antibody-dependent cellular cytotoxicity in which the pathogen does not need to be phagocytosed to be destroyed. During this process, the pathogen is opsonized and bound with the antibody IgG. The antibody triggers a release of lysis products from cells like monocytes, neutrophils, eosinophils, and natural killer cells. This process can cause inflammation of surrounding tissues and damage to healthy cells. (Antibody Opsonization)
[00102] "P24 Antigen Assay". Viral isolation through viral culture, nucleic acid tests to detect viral RNA, and tests to detect p24 antigen can be used to demonstrate virus or viral components in blood, thereby verifying infection. The p24 antigen assay measures the viral capsid (core) p24 protein in blood that is detectable earlier than HIV antibody during acute infection. p24 antigen is found in serum in either free form or bound by anti-p24 antibody. Free p24 can be measured with enzyme immunoassays whereas detection of bound p24 requires pretreatment with an acid to dissociate the complex. Although procedures vary between manufacturers, HIV p24 antigen tests employ ELISA technology with modifications to detect antigen, not antibody. In a representative assay, such as an "antibody sandwich" type, a specific monoclonal antibody to HIV p24 is attached to the solid phase (microtiter plate-well or polystyrene bead) acting to "capture" the viral antigen in the sample when added. The sample is diluted in a Triton X100 detergent to disrupt virions, and if antigen is present in the serum, the antigen will attach to the monoclonal antibody on the solid phase. Following a wash step, an antibody detector is added and incubated. This detector reagent is usually a high-titer antibody to p24 antigen that is coupled to biotin. Subsequently, incubation with a conjugate (streptavidin-peroxidase) labels the complex by attaching via biotin. An avidin-biotin system acts as an amplifier to generate additional signal to detect the small quantities of antigen in the sample. Addition of a substrate (tetramethylbenzidine) will allow the production of color as the enzyme cleaves the substrate. A weak acid (e.g., 2 M sulfuric) is finally added to stop the reaction after a defined period of time. Resultant optical density values are proportional to the amount of HIV-1 p24 antigen in the specimen. This assay can detect p24 antigen in the pg/ml-to-ng/ml range. The optical density is read with a spectrophotometer at 450 nm. A protocol for an in-house p24 antigen assay designed for testing large numbers has been described and is more cost effective than commercially available assays. (27 Mckeating J. Quantitative assays for virus neutralization. In: Karn J, ed. HIV: A Practical Approach, Virology and Immunology, vol 1. Oxford: IRL Press at Oxford University Press, 1995;118-127.)
[00103] To determine the levels of p24 antigen in blood, an HIV-1 antigen standard is diluted to prepare a series of six standards of varying concentrations. Concentrations vary between 0.0 and 125 pg/ml. A standard curve is generated from which optical density values of the unknown specimens are interpolated to determine their concentration. The standard curve is constructed using a linear graph and plotting the concentration of the HIV-p24 antigen standard (pg/ml) on the X-axis versus the mean optical densities for each standard on the Y-axis. Each standard is added in duplicate wells, and at least 5 controls must be included (3 negatives and 2 positives). If the value of the unknown sample is higher than the value of the highest standard, the sample must be diluted in normal human serum and the entire neutralization procedure is repeated.
[00104] To improve sensitivity of the p24 antigen assay, an Immune Complex Dissociation (ICD) procedure may be introduced using low pH to dissociate p24 antigen/anti-p24antibody complexes before performing the antigen assay. Using this procedure, an increased sensitivity of the assay was demonstrated, particularly for asymptomatic HIV-infected individuals. This dissociation procedure allows for detection of both free p24 antigen and complexed p24 antigen/antibody. The method not only increases the number of antigen positive individuals (epidemiologic sensitivity), but also can detect lower amounts of p24 antigen (analytical sensitivity). The sensitivity of this assay may be further enhanced using a signal amplifying step that involves the addition of a tyramide compound which generates an intermediate to produce more enzyme and substrate molecules; and hence, more signal.
[00105] A "Peripheral Blood Mononuclear Cell" (PBMC) is a blood cell having a round nucleus, such as a lymphocyte or a monocyte. These blood cells are a critical component in the immune system to fight infection and adapt to intruders. The lymphocyte population consists of T cells (CD4 and CD8 positive -75%), B cells and NK cells (-25% combined). These cells are often extracted from whole blood using ficoll, a hydrophilic polysaccharide that separates layers of blood, with monocytes and lymphocytes forming a buffy coat under a layer of plasma. This buffy coat contains the PBMCs. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis which will preferentially lyse red blood cells. This method results in neutrophils and other polymorphonuclear (PMN) cells which are important in innate immune defense to be obtained. PBMCs are widely used in research and clinical uses every day. HIV research uses them because PBMCs include CD4+ cells, the cells HIV infects.
[00106] Peripheral Blood Mononuclear Cells (PBMC) Assay PBMC were prepared by Percoll centrifugation and depleted of CD8 cells with anti-CD8 antibody-coated magnetic beads (Dynabeads, DYNAL). Virus isolation was carried out by coculture of PBMC from infected and normal individuals after stimulation with anti-CD3 antibody (clone CLB-CD3). PBMC grown for 4 days were infected with the virus inoculum and incubated for 3 days in culture medium containing serially decreasing concentrations of antiretroviral drugs. The p24 concentrations in culture supernatant were measured with mini VIDAS (bioMerieux) and the 50% and 90% inhibition concentrations (IC50 and IC90) were obtained from the dose-response curve. The antiretroviral drugs being assayed were AZT, ddl, 3TC, d4T, ABC, NVP, EFV, IDV, SQV, NFV, APV, and LPV. The HIV-1 LAI isolate was used as reference. RESULTS: Virus isolation was succeeded from infected individuals with viral loads of 400 copies/ml or more (n = 60). In control experiments using the LAI isolate, the average CV values for all drugs were 29% for IC50 and 18% for IC90. Based on these data, resistance was defined as the 4-fold increase in IC50 and IC90. So far, nine drug-naive and seven drug-experienced patients were assayed. Drug-resistant virus was not observed in any drug-naive patient, and found in five drug-experienced patients. The drug-resistant viruses observed showed cross-resistance to 12, 10, 9, 9, and 3 different drugs. CONCLUSIONS: The phenotypic assay presented are able to assess drug susceptibility in PBMC with a low standard errors. This may be useful for evaluation of the role of drug resistance in lymphocytes in vivo. A standardized antiviral drug susceptibility assay for clinical human immunodeficiency virus type 1 (HIV-1 ) isolates has been developed for use in clinical trials. The protocol is a two-step procedure that first involves cocultivation of patient infected peripheral blood mononuclear cells (PBMC) with seirnegative phytohemagglutinin-stimulated donor PBMC to obtain an HIV-1 stock. The virus stock is titrated for viral infectivity (50% tissue culture infective dose) by use of serial fourfold virus dilutions in donor PBMC. A standardized inoculum of 1 ,000 50% tissue culture infective doses per 106 cells is used in the second step of the procedure to acutely infect seronegative donor PBMC in a 7-day microtiter plate assay with triplicate wells containing zidovudine (ZDV) concentrations ranging from 0 to 5.0 μM.
[00107] The conventional PBMC based assay [59-61] with readout based on p24 antigen production involves multiple rounds of virus replication, has a moderate reproducibility and sensitivity, is time-consuming and cumbersome to perform but involves the most physiological target cell. An alternative readout can be the measurement of viral RNA, which shortens the time by several days [62]. Intracellular (IC) p24 antigen determination in infected PBMC cultures may be run as a single round assay with increased sensitivity, reproducibility and speed but it is not easy to perform [63]. The method of measuring ICp24 was also applied to other target cells, like macrophages [64]. Plaque reduction assays use either U87.CD4 or GHOST(3) cells engineered to express coreceptors for HIV [65]. In U87.CD4 cells the syncytium- inducing capacity of HIV is exploited, while infected GHOST(3) cells turn green due to the activation of the GFP gene linked to the HIV-2 LTR. These assays are single round, highly reproducible, easy to perform, with sensitivity comparable to the PBMC assay, but require a shorter time. The fusion assay is based on fusion of effector cells expressing the native HIV-1 envelope on their surface (PM 1 persistently infected with HIV-1 ) with target cells expressing the appropriate receptors (initially NIH-3T3 mouse fibroblasts or HeLa human epithelial cells stably expressing human CD4, CCR5 and/or CXCR4). The readout is measurement of β-galactosidase activity [66]. Pseudovirus (PSV)-based assays exist in a number of variant assay formats using different target cells [67, 68]. A selected molecular clone is tested in a single round assay with luciferase readout that results in short-term assays with high reproducibility and sensitivity. Plasmid production and producer cell line culture history are crucial criteria and influences the results. Due to this a fairly large inter-laboratory variation has been documented [41]. Finally, assays using recombinant viruses have also been included [69]. This assay type was run with two different starting materials, env sequences were amplified either from culture supernatants or from cloned plasmid.
[00108] "Primary isolates of HIV-1 ": These are isolates of HIV-1 that are found spontaneously in human populations. Commonly, such isolates are obtained from clinical specimens taken from individuals naturally infected with HIV-1. Primary isolates differ from latoratory isolates in that the latter are strains of HIV-1 that are adapted to growth in transformed T cell lines.
[00109] The various "signal generating compounds" (labels) contemplated include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, achdiniums, phenanthridiniums and luminol, radioactive elements, and direct visual labels. Examples of enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
[00110] Solid phases" ("solid supports") are known to those in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes (stabilized human red cells) and others. The "solid phase" is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like. A "solid phase", as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or nonmagnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes and other configurations known to those of ordinary skill in the art.
[00111] It is contemplated and within the scope of the invention that the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens. Microporous structures are generally preferred, but materials with gel structure in the hydrated state may be used as well. These materials may be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics.
[00112] Other embodiments which utilize various other solid phases also are contemplated and are within the scope of this invention. For example, ion capture procedures for immobilizing an immobilizable reaction complex with a negatively charged polymer. An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged polyanion/immune complex and the previously treated, positively charged porous matrix and detected by using various signal generating systems previously described, including chemilumninescent signal measurements.
[00113] Also, the methods of the present invention can be adapted for use in systems which utilize microparticle technology including in automated and semi-automated systems wherein the solid phase comprises a microparticle (magnetic or non-magnetic).
[00114] The use of scanning probe microscopy (SPM) for immunoassays also is a technology to which the peptides of the present invention are easily adaptable. In scanning probe microscopy, in particular in atomic force microscopy, the capture phase, for example, at least one of the peptides disclosed herein, is adhered to a solid phase, a test sample suspected of containing the antibody of interest is contacted with the solid phase and a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase. The use of scanning tunnelling microscopy eliminates the need for labels which normally must be utilized in many immunoassay systems to detect antigen/antibody complexes. The use of SPM to monitor specific binding reactions can occur in many ways. In one way, one member of a specific binding partner is attached to a surface suitable for scanning. The attachment of the analyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art. Or, covalent attachment of a specific binding partner (analyte specific substance) to a test piece which test piece comprises a solid phase of dehvatized plastic, metal, silicon, or glass may be utilized. Covalent attachment methods are known to those skilled in the art. Also, polyelectrolyte interactions may be used to immobilize a specific binding partner on a surface of a test piece by using techniques and chemistries. Following attachment of a specific binding member, the surface may be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding. The surface also may be scanned either at the site of manufacture or point of use to verify its suitability for assay purposes. The scanning process is not thought to alter the specific binding properties of the test piece.
[00115] A "specific binding member," as used herein, is a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog. Immunoreactive specific binding of folate-binding protein to determine folic acid, or the use of a lectin as a member of a specific binding pair for the determination of a carbohydrate. The specific binding pair member can include a protein, a peptide, an amino acid, a nucleotide target, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog. Immunoreactive specific binding members include antigens, antigen fragments, antibodies and antibody fragments, both monoclonal and polyclonal, and complexes thereof, including those formed by recombinant DNA molecules.
[00116] The term "test sample" refers to a component of an individual's body which is the source of the analyte (such as, antibodies of interest or antigens of interest). These components are well-known in the art. These test samples include biological samples which can be tested by the methods described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; and biological fluids such as cell culture supernatants; fixed tissue specimens; and fixed cell specimens.
Detailed HIV Structure and Active Pathway
[00117] The interactions of HIV-1 for fusion with the plasma membrane lipid bilayer lipids are illustrated in FIG. 5. Plama membrane glycosphingolipid microdomains as preferential sites of formation of the HIV-1 fusion complex. In the plasma membrane of CD4+cells, CD4 1 is present in glycoshingolipid enriched microdomains but is not associated with HIV-1 coreceptors. Once bound to CD4, the viral particle 2 is conveyed to an appropriate coreceptor 3 by the glycosphingolipid raft 4, which moves freely in the external leaflet of the plasma membrane 5, cholesterol 6; glycosphigolipid 4; phosphatidylcholine 7. [00118] As shown in FIG. 1 , after budding from host cells, the HIV-1 virus 2 exhibits a strong tendency to infect T lymphocytes as target cells, using CD4 1 as a receptor (Piguet & Sattentau 2004). FIG. 1A is a cross section of the HIV-1 envelope protein. The binding and fusion of HIV with the target cell involves a choreographed ballet between the proteins of the free virus 2 and the entry site of the target cell (FIGS. 1 B and 1C). HIV entry into a cell is a multistep process initially involving the interactions of viral envelope protein gp120 and gp41 with several binding sites on the cell surface. The envelope proteins exist as a trimer consisting of 3 gp120 molecules and 3 gp41 molecules. The binding of pg120 to CD3 is followed by conformational changes in the gp120 protein that expose binding sites to chemokine receptors 3, CXCR4 or CCR5, that serve as co-receptor binding sites for interactions of the virus with the target cell (Berger ct al., 1999; Doms, 2000; Huang et al 2005). The binding of gp120 to the chemokine co-receptor, in turn, induces conformational changes that allow the binding of the gp41 anchor protein to the cell, and this is followed by fusion of the viral lipid bilayer with the plasma membrane bilayer, and entry of the virion RNA into the target cell (Colman & Lawrence, 2003) (FIG. 1C). The binding and entry processes entail numerous types interactions between proteins and lipids of the virus and specific lipids of the target cell (Fantini et al. 2002).
[00119] In FIG. 1 , the reference numbers represented as follows CD4 1 , viral particle 2, co-receptor 3, raft 4, plasma membrane 5, p17 matrix 10, lipid bilayer 11 , membrane proximal region 18, fusion peptide 19. The arrow represents step 3 of fusion and entry. [00120] To achieve a vaccine that would cover numerous clades highly conserved sequences are the preferred focus. Two such conserved antigenic regions are portions of the Membrane Proximal Region (MPR) of gp41 and the lipid bilayer itself, including lipids such as phosphatidylinositol phosphate, phosphatidylserine, phosphatidylglycerol, and cholesterol. Additionally, certain conserved regions in gp120, particularly conserved elements of the V3 loop, are known to bind to glycolipids, including galactosyl ceramide and ganglioside GM3. The MPR region 18 of gp41 as shown in FIG. 6 contains the binding epitopes for two human IgG monoclonal antibodies that are known to be broadly neutralizing antibodies. They are known as 2F5 and 4E10. 2F5 binds to ELDKWA (the MPR starts at D) and 4E10 binds to NWFDIT. The 2F5 epitope, ELDKWA, is the same sequence identified as the binding site for GalCer. Both the 2F5 and 4E10 epitopes on gp41 are shown as labeled on FIG. 7. The cholesterol binding site LWYIK is at the end of the MPR. The overall series of interactions of HIV-1 involving budding, binding and fusion with host and target cells exposes lipid-associated proteins, and even lipids themselves, as targets for virus neutralization.
[00121] FIG. 7 is a schematic model of the HIV-1 putative trimehc envelope spike. The viral particle 2 is shown inserted into the plasma membrane 5. Most of the surface of gp41 is believed to be occluded by gp120. However, the amino acid sequences of gp41 close to the membrane that have been identified as binding sites of MABs 2F5, Z13, and 4E10 have been suggested to be exposed to antibody binding [70]. IgG is shown as 20. Carrier Neutralizing Assay Procedure
[00122] In this method of assay, the HIV-1 virus is allowed to bind to a carrier in such a manner that the carrier-bound HIV-1 retains the ability to infect a target cell. Prior to such an infection the carrier-bound HIV-1 is exposed to candidate neutralizing antibodies. The unbound candidate neutralizing antibodies are then removed by washing the carrier free of unbound antibodies. As an example, if a cell lacking CD4 (a CD4-negative cell) were to serve as a carrier for binding to HIV-1 , and if the CD4- negative cell could not be infected by the HIV-1 , the HIV-1 would be retained on the surface of the cell where it could be tested for binding of antibody and the unbound antibody could be washed off by a variety of standard laboratory methods, such as simple centhfugation, or other methods of separating cells from the surrounding liquid milieu.
Interactions of HIV-1 with Cd4-Negative Cells
[00123] HIV-1 binds to CD4-negative cells through several mechanisms. One way is through mediation of antibodies binding to HIV-1 to form immune complexes, and the immune complexes binding to complement receptor type I (CR1 ) on erythrocytes. This is a well-known mechanism for removal of immune complexes from blood, and is a mechanism that involves HIV-1 [71 , 72]. Another mechanism is through binding of HIV- 1 to Duffy blood group antigen [73]. Yet another mechanism is through direct binding of HIV-1 to CD4-negative cells in the circulation [74]. It has been experimentally demonstrated that both a laboratory strain (MN) and two primary isolates of HIV-1 (TH and GP) each bound to a variety of CD4-negative cells, including Raji cells (B lymphocytes), erythrocytes, platelets, and neutrophiles.
[00124] According to dinger et al. [74], HIV-1 that is bound to CD4-negative cells, such as Raji cells (B lymphocytes), erythrocytes, platelets, neutrophiles, etc., is far more infectious to CD4-positive T cells than free virus (17 times more infectious when HIV-is bound to Raji cells, and 2-3 times more infectious when HIV-1 is bound to erythrocytes or platelets). It was proposed that the infectivity of CD4-positive cells occurred through cell-to-cell contact. Presumably, the CD4 binding had the higher affinity for HIV-1 binding, thus promoting transfer of the HIV-1 to the CD4-positive cells (see Table 1 from dinger et al., [74] incorporated by reference in its entirety herein).
[00125] dinger et al do not speculate on the mechanism of binding of HIV-1 to
CD4-negative cells, but it seems reasonable to presume that binding could have occurred to the glycosphingolipids on the cell surfaces, such as GalCer, CTH, GM3, or other similar lipids that bind HIV-1 [74]. The glycosphingolipids might have become exposed in damaged or senescent erythrocytes, and such cells are known to express simple glycosphingolipids (such as alpha-GalCer) that are thought to allow binding of natural antibodies (such as antibodies that recognize glycosphingolipids that have a terminal alpha-Gal) for removal of senescent erythrocytes [75]. CTH is an example of a glycolipid with a terminal alpha galactose. [00126] Lipid bilayer lipids are cryptic antigens in normal cells, and are protected from binding of antibodies to lipids by abundant overlying membrane proteins or glycoproteins. This is dramatically illustrated by the observation that large numbers of B cells that secrete IgM or IgG antibodies to phosphatidylcholine are easily demonstrated in mice, rats, rabbits, and humans [76-78]. These antibodies are capable of binding to phosphatidylcholine, or to liposomes containing [79, 80], but binding to phosphatidylcholine on erythrocytes occurs only when the erythrocytes have been treated with a proteolytic enzyme (bromelin) [76-78, 80-82]. Erythrocytes that are aged have reduced amounts of sialic acid that is thought to allow removal of senescent cells [83], and this might promote increased visibility of the underlying lipids.
[00127] Based on all of the above, it seems reasonable to believe that underlying lipids that might bind HIV-1 , such as GalCer, CTH, and other widespread cellular glycolipids, might be responsible for binding of HIV-1 to CD4-negative cells. It is also reasonable to believe that if such cells should bind HIV-1 , the cells would not themselves be infected by HIV-1. It is further reasonable to believe that the HIV-1 that is bound to the CD4-negative carriers cells would have infectivity for CD4-positive cells that is at least as strong, and perhaps stronger, than infectivity by free virus.
General Carrier Neutralization Assay Protocol
[00128] I proposed that HIV-1 that is circulating in the blood becomes bound to circulating CD4-negative elements of the blood that serve as carriers that cannot be infected (see above). Since the bound virus has greatly increased infectivity for CD4- positive T lymphocytes when compared to free virus, a new type of neutralization assay could be developed by pre-treatment of the CD4-negative cell-HIV-1 complex with presumptive neutralizing antibodies. The unbound antibodies could be removed (for example after binding to erythrocytes) by centrifugation or other types of washing, and the washed cells would be co-cultured with CD4-positive target cells (for example, peripheral blood mononuclear cells or H9 T cell line) as an indicator for whether the antibodies neutralized the carrier-bound HIV-1. For each assay run, the positive control would consist of the CD4-negative cell-HIV-1 complex that would exhibit the expected level of infectivity of co-cultured CD4-positive cells.
[00129] If erythrocytes are used as CD4-negative carriers, the cells themselves could be examined for the effects of antibody-mediated complement activation in the presence of the bound antibodies by hemolyzing the cells in a standard complement lysis assay.
[00130] The following examples illustrate the characterization and uses of the various embodiments of the invention. These examples are illustrative only and do not limit the scope of the invention.
Example I Duffy Antigen Receptor Protein as Carrier [00131] The Duffy antigen is a protein located on the surface of red blood cells and is named after the patient in which it was discovered. In humans, this protein is encoded by the DARC gene. The protein encoded by this gene is a glycosylated membrane protein and a non-specific receptor for several chemokines. The protein is also the receptor for the human malarial parasites Plasmodium vivax and Plasmodium knowlesi. Polymorphisms in this gene are the basis of the Duffy blood group system. The gene is also known as CCBP1 , Glycoprotein D (GPD), Dfy and CD234.
[00132] The Duffy Antigen Receptor for Chemokines (DARC) belongs to a family of erythrocyte chemokine receptors that bind C-X-C and C-C chemokines such as interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1 ) and regulated-on- activation, normal T cell-expressed and -secreted (RANTES), but not macrophage inflammatory protein 1 alpha (MIP-1 alpha) or MIP-1 beta. DARC has also been identified to a receptor for malaria parasites Plasmodium vivax and Plasmodium knowlesi. In the present study, we show that HIV-1 binds to RBCs from Caucasian individuals via DARC making RBCs able to transmit HIV to peripheral blood mononuclear cells (PBMCs). RBCs may function as a reservoir for HIV-1 or as a receptor for the entry of HIV-1 into CD4-cell subsets as well as neurons or endothelial cells. HIV-1 attaches to RBCs via DARC, effecting trans-infection of target cells.
Purifying Duffy antigens in human erythrocytes [00133] Using the anti-Fy6 monoclonal antibody, follow a procedure for purification of Duffy antigens in human erythrocytes.
Materials
[00134] Human blood was obtained from our institution and hemagglutination tests were performed according to standard procedures. Goat anti-mouse IgG horseradish peroxidase conjugate was obtained from Bio-Rad. Anti-mouse IgG-Sepharose beads were obtained from Cappel. Protein A-Sepharose CL-4B beads were obtained from Pharmacia LKB Biotechnology Inc. The murine monoclonal anti-Fy6 antibody was obtained and purified as described elsewhere [84]. A murine monoclonal antibody which reacts specifically with type M glycophorin A was produced as described [85]. A murine monoclonal antibody which reacts specifically with type N glyco-Fhorin A and B and rabbit antibody which reacts with types MN glycophorin A were supplied by Dr. 0. Blumenfeld (Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY) (10). Monoclonal antibody R1.3 which reacts with both glycophorin A and B was supplied by Dr. D. J. Anstee (South Western Regional Transfusion Center, Bristol, United Kingdom). Rabbit polyclonal antibody to human Band 3 protein was supplied by Dr. V. Marchesi (Department of Pathology, Yale University School of Medicine). Rabbit antibody to actin was supplied by Dr. D. Fischman (Department of Cell Biology/Anatomy Cornell University Medical College). Ultrapure urea was obtained from Schwarz/Mann. Chymotrypsin was obtained from Boehringer Mannheim. SDS (sequencing grade) was obtained from Calbiochem. Triton X-100, Tween 20, and Hepes were from Sigma. Monoclonal Antibody Binding to Red Cells
[00135] Red cells were washed three times in cold PBS (pH 7.4), resuspended in the same solution, and mixed continuously overnight at 4 0C with anti-Fy6 antibody at a concentration of 10 μg/ml of packed red cells. This concentration, determined with radioiodinated antibody, exceeds the concentration required to saturate Duffy antigen sites. Unbound antibody was removed by washing the red cells with cold PBS. Red cell ghosts were prepared by hypotonic lysis with 20 volumes of cold 5 mM sodium phosphate buffer (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride and 100 kallikrein-inactivating units/ml Trasylol (aprotinin). Then the ghosts were washed exhaustively until they were light pink in color. Ghosts were centhfuged for 30 min at 43,000 X g; supernatant was decanted, and the pellet was made to 50 mM Hepes- NaOH, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 100 kallikrein-inactivating units/ml Trasylol, and frozen at -20 0C. Frozen ghosts prepared in this way can be stored for months without loss of Duffy antigens.
Preparation of a Detergent-soluble Erythrocyte Membrane Fraction
[00136] Frozen ghosts were thawed and centhfuged for 30 min a t 43,000 X g. The pellet was resuspended in 50 mM Hepes-NaOH, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 100 kallikrein-inactivating units/ml Trasylol to three times the initial volume of packed red cells. Triton X-100 (peroxide-free) was added to a final concentration of 1 %, and the solution was mixed gently for 1 h at room temperature. Shells were removed by centrifugation for 30 min at 43,000 X g. The supernatant was concentrated 4-fold in an Amicon concentrator using a PM Y10 filter (Amicon Corp.) under nitrogen pressure. Purification of Antigen-Antibody Complex
[00137] A 0.1 volume of PBS solution, 10 times the normal concentration, was added to the detergent extract. The detergent extract was then incubated with Sepharose 4B beads coupled to anti-mouse IgG for 1 h at room temperature. The ratio of beads to detergent extract was 1 :100 (v/v). The antimouse IgG-Sepharose beads were removed by centrifugation, and washed in a solution containing PBS and 0.5% Triton X-100 at a 1 :20 (v/v) ratio of beads to washing solution. The washings were done at room temperature for 15 min and repeated three times. Elution was done by incubating the beads in a solution containing 62.5 mM Ths-HCL (pH 6.8), 0.5% SDS at a 1 :2 (v/v) ratio beads to eluant. The incubation was at 65 0C for 10 min and repeated three times. The eluted material was concentrated in an Amicon concentrator with PM Y10 filter (Amicon Corp.) under nitrogen pressure.
Cloning Duffy ntigens
Partial Amino Acid Sequence Analysis.
[00138] The purified protein from Fy(a-b+) human erythrocytes was alkylated and cleaved with cyanogen bromide (CNBr) as explained [86]. Pe- 1 peptide was obtained by sequencing the nonfractionated CNBr digest using the o-phthalaldehyde blocking reagent [84]. Pe-5 peptide was the partial sequence of the only fragment (-4 kDa) that separated very well from the CNBr digest run on the threelayer SDS/PAGE system [87].. After the run, the peptide fragment was electroblotted onto ProBlott (Applied Biosystems) and sequenced [84]. Another aliquot was digested with pepsin (50: 1 ratio) at 37°C overnight, and the fragments were separated by reverse-phase HPLC using a Vydac Ci8 column. Pe-2, Pe-3, Pe-4, and Pe-6 peptides, which were the few pepsin peptides yielded by reverse-phase HPLC, were sequenced. Applied Biosystems protein/peptide sequencer, model 470 or 477, was used according to the manufacturer's recommendations.
[00139] Primer Design and PCR. The nucleotide sequence of the primers (23-mer each) was deduced from the N-terminal and C-terminal amino acid sequences of Pe-5 peptide. Bases were chosen according to the codon preference described by Lathe (10), and deoxyinosine (I) was incorporated at the position where degeneracy exceeded >3-fold, except toward the 3' end. Primer A (sense) was specific for residues 245-252 and consisted of 12-fold degeneracy δ'-ATGAAYATHYTITGGGCITGGTT (where Y = C or T; and H = C, T, or A). Primer B (antisense) was specific for residues 261 -268 and consisted of 32-fold degeneracy 5'-ACIAGRAARTCIAGICCIARNAC (where R = A or G; and N = G, A, T, or C) [88].
[00140] First-strand cDNA was synthesized from Fy(a-b+) phenotype mRNA using the preamplification kit from BRL and oligo(dT) as primer. For enzymatic amplification, cDNA, primer A, primer B, and Taq polymerase (Stratagene) were incubated in a Perkin-Elmer thermal DNA cycler. The amplification product of expected size (72 bp) was subcloned in pBluescript-SK vector (Stratagene). The deduced amino acid sequence of the insert matched the sequence of Pe-5 peptide. From the sequence WFIFWWPH of peptide Pe-5, the oligonucleotide TGGTTTATTTTCTGGTGGCCTCAT [00141] was chemically synthesized, 32P labeled at the 5' end with T4 polynucleotide kinase (New England Biolabs), and used as a probe to screen a human bone marrow cDNA library [88].
Human mRNA and DNA Isolation.
[00142] PoIy(A)+ RNA was isolated as explained [89] and by using the Invitrogen isolation kit. mRNAs from Caucasian adult liver, spleen, kidney, brain, and fetal liver, as well as erythroleukemia cells K-562, were obtained from Clontech. DNA was obtained from peripheral blood leukocytes of the four Duffy phenotypes by a slight modification of a published procedure [90].
RNA-Blot Analysis (Northern).
[00143] PoIy(A)+ RNAs were run on formaldehyde/agarose gel and transferred onto Hybond-N+ nylon membranes (Amersham). They were hybridized in QuickHyb (Stratagene) and washed according to the manufacturer's instructions.
DNA-Blot Analysis (Southern).
[00144] All restriction enzyme digestions were done according to the conditions suggested by the supplier (New England Biolabs). Digested DNA was size-fractionated on 0.8% agarose gel and blotted as described for Northern analysis. Hybridization in QuickHyb solution was carried out at 680C for 1 hr according to the manufacturer's instructions.
Construction
[00145] A mixture of mRNA of several Fy(a-b+) individuals, the BRL Superscript Choice system, and oligo(dT) as a primer was used to prepare cDNA. The cDNA was ligated into λZAP Il vector and packaged with Gigapack Gold (Stratagene) extract. About 1.9 x 106 unamplified cDNA clones were screened with the 32P-labeled probe described above. cDNA inserts in pBluescript were isolated by the plasmid-rescue method according to manufacturer's protocol. Both DNA strands were sequenced by using vector primers and by primers designed from the sequenced regions of the transcript [88].
Primer Extension.
[00146] A 32P-labeled 24-mer antisense primer from nt 57-80 of the coding strand was extended on Fy(a-b+) mRNA using a preamplification kit (BRL), and the products were separated on a 6% sequencing gel.
Incubation with HIV-1
[00147] The DARC protein, whether cloned or purified from human erythrocytes is then incubated with HIV-1 by means known in the art. DARC-Bound HIV-1 Neutralization Assay
[00148] The carrier-bound HIV-1 is exposed to candidate neutralizing antibodies. The unbound candidate neutralizing antibodies are then removed by washing the carrier free of unbound antibodies. As an example, if a cell lacking CD4 (a CD4-negative cell) were to serve as a carrier for binding to HIV-1 , and if the CD4-negative cell could not be infected by the HIV-1 , the HIV-1 would be retained on the surface of the cell where it could be tested for binding of antibody and the unbound antibody could be washed off by a variety of standard laboratory methods, such as simple centrifugation, or other methods of separating cells from the surrounding liquid milieu. The level of viral activity and concomitant inhibition are then measured by various means known in the art and described herein.
Example 2
Indirect Binding of HIV-1 To Erythrocytes Through Cells Immune Complexes
[00150] One obvious way is through mediation of antibodies binding to HIV-1 to form immune complexes, and the immune complexes binding to complement receptor type I (CR1 ) on erythrocytes.
[00151] Immune complexes (IC)3 formed by HIV/anti-HIV Abs (HIV-IC) are present in plasma and can bind specific FcRs [91 , 92]. Complement activation followed by C3 fragment deposition on the HIV-IC allows efficient binding of HIV to cells such as B lymphocytes, which have receptors for C3 fragments, in particular CD35 and CD21 [93]. CD35, also called complement receptor 1 (CR1 ), binds C3b and C3bi and is expressed on erythrocytes as well [72].
Materials and Methods
Virus purification
[00152] HIV used in all experiments was of HIV-1 type IMB of the North American isolate, cultured on Hut/4-3 cells, a cell line derived from human lymphocytes. Cell supernatants containing the virions were inactivated by incubation for 1 h at 62°C in the presence of 0.02% formaldehyde to preserve the antigenic properties of the virions. Supernatants were separated from cell debris by low speed centrifugation at 400 x g for 15 min and passed through a 0.45-μm pore size membrane filter (Minisart filter; Sartorius, Gottingen, Germany). The viral suspension was further concentrated using Centhprep centrifugal filter devices (Mr 10,000 cut-off; Millipore, Bedford, MA), the concentrate was ultracentrifuged twice for 1 h each time at 160,000 x g, and the pellet was resuspended in PBS. HIV-1 was purified by gel filtration on a Sephadex G-25 column (Pharmacia Biotech, Uppsala, Sweden). The HIV-1 was stored at - 800C until use [72].
Erythrocytes
[00153] Human erythrocytes were purified from fresh blood of healthy volunteers with blood group O using dextran sedimentation. Briefly, the blood was centrifuged at 680 x g for 7 min, and plasma and buffy coat were removed. Remaining blood cells were mixed with 4% dextran and PBS and left on ice for 40 min. After sedimentation, the supernatant was removed with the upper layer of the erythrocytes, and the rest of the purified RBC were washed five times with RPMI 1640 medium (Life Technologies, Basel, Switzerland), each time removing the upper layer of the cells with the supernatant. The final contamination with leukocytes was reduced to < 1 leukocyte/10,000 RBC [72].
IC formation and purification
[00154] HIV/anti-HIV-IC were formed by incubating ~ 1.5 μg of HIV-1 with 85 μg of anti-HIV Ig anti-human IgG (Southern Biotechnology Associates, Birmingham, AL) in a total volume of 220 μl in a shaking water bath for 60 min at 37°C. IC opsonization with complement was achieved by incubation with either fresh NHS (dilution, 1/3) or other complement-deficient sera for 7 min at 37°C. The opsonized HIV-IC were purified by sucrose density gradient ultracentrifugation, as previously described. In short, sucrose gradients consisted of five layers of sucrose solutions 50 to 10% in PBS. Opsonized HIV-IC or HIV were overlaid on the gradients and ultracentrifuged for 2.5 h at 116,000 x g. Fractions of 200 μl were taken from the bottom of the tube, and the cpm of each fraction were determined. Fractions containing purified opsonized HIV or HIV-IC, located near the bottom of the gradient, were then incubated with erythrocytes [72].
Binding of HIV-IC or HIV to Erythrocytes
[00155] Purified opsonized HIV-IC or HIV were incubated with erythrocytes at a ratio of 6000 cpm of the opsonized HIV-IC or HIV/2 x 108 erythrocytes for 10 min at 37°C. The experimental conditions were established to correspond to the situation in vivo. In vivo, the number of viruses are up to 100,000 copies/ml blood, i.e., one virus for 104 erythrocytes. After incubation, the reaction was stopped by adding ice-cold RPMI 1640 and was centrifuged for 3 min at 170 x g. A fraction corresponding to half the final volume was removed, and the pellet of erythrocytes was resuspended in the remaining half [72].
Incubation with Candidate Neutralizing Antibodies
[00156] The carrier-bound HIV-1 is exposed to candidate neutralizing antibodies. The unbound candidate neutralizing antibodies are then removed by washing the carrier free of unbound antibodies. As an example, if a cell lacking CD4 (a CD4-negative cell) were to serve as a carrier for binding to HIV-1 , and if the CD4-negative cell could not be infected by the HIV-1 , the HIV-1 would be retained on the surface of the cell where it could be tested for binding of antibody and the unbound antibody could be washed off by a variety of standard laboratory methods, such as simple centrifugation, or other methods of separating cells from the surrounding liquid milieu. The washed cells are then co-cultured with CD-4 positive target cells (for example, peripheral blood mononuclear cells or H9 T cell lines) to be subsequently subjected to the neutralizing assay. For each assay run, the positive control would consist of the CD4-negative cell- HIV-1 complex that would exhibit the expected level of infectivity of co-cultured CD4- positive cells.
Complement Lysis Assay
[00157] If erythrocytes are used as CD4-negative carriers, the cells themselves could be examined for the effects of antibody-mediated complement activation in the presence of the bound antibodies by hemolyzing the cells in a standard complement lysis assay.
[00158] Nonnucleated cells, such as erythrocytes, lyse with relative ease when exposed to sensitizing antibody and complement. In the case of erythrocytes, this causes release of hemoglobin, which can easily be measured in the supernatant. Simple, direct and automoation-ready procedures for measuring hemoglobin concentration are readily available, such as the QUANTICHROM TM hemoglobin assay kit manufactured by BioAssay Systems (Harward, CA). This system features a "mix- and-read" procedure that involves the addition of a single working reagent and reading the optical density which is easily automated for high-throughput assay. The method utilizes a Triton/NaOH method whereby the hemoglobin is converted into a uniform colored end product. The intensity of color, measured at 400 nm, is directly proportional to hemoglobin concentration in the sample. In comparison, measuring the lysis of living nucleated cells is more challenging. Nucleated cells, unlike erythrocytes, do not contain a natural chromophore. Nucleated cells have active budding processes to shed membrane attack complexes (MAC) from the membrane and they have ion-pumps that actively reclaim released ions, stabilizing the osmotic balance and preventing rupture of the plasma membrane.
Example 3 Direct Binding of HIV-1 to Cd4-Negative Cells
[00159] Virus bound to CD4- cells was up to 17 times more infectious for T cells in cocultures than was the same amount of cell-free virus. The enhanced infection of T cells by virus bound to CD42 cells was not due to stimulatory signals provided by CD42 cells or infection of CD42 cells. However, anti-CD18 antibody substantially reduced the enhanced virus replication in T cells, suggesting that virus that bound to the surface of CD42 cells is efficiently passed to CD41 T cells during cell-cell adhesion. These studies show that HIV binds at relatively high levels to CD42 cells and, once bound, is highly infectious for T cells. This suggests that virus binding to the surface of CD42 cells is an important route for infection of T cells in vivo. Most CD42 cells do not support virus replication, some have speculated that HIV binding to uninfectable cells could provide a mechanism for clearance of virus from circulation [74].
Materials and Methods
Cell lines and isolation of primary cells.
[00160] The T-lymphocytic H9 (HTB-176) and B-lymphocytic Raji (CCL-86) cell lines used were obtained from the American Type Culture Collection (ATCC; Manassas, Va.). Cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Whittaker M. A. Bioproducts, Walkersville, Md.) and gentamicin (Sigma, St. Louis, Mo.) at 50 mg/ml. Antibodies to leukocyte function-associated antigen type 1 b (LFA-I b; CD18) and CD14 were obtained from the TS1/18.1.2.11 hybridoma (HB- 203; ATCC) and the 261 C hybridoma (HB-246; ATCC), respectively. Each antibody was purified using an Affinity Pak Immobilized Protein A column (Pierce, Rockford, III.) [74].
[00161] PBMC obtained from healthy donors were isolated by Ficoll-Hypaque gradient centhfugation (Whittaker M. A. Bioproducts). Stimulated PBMC were produced by culture in medium containing phytohemagglutinin (PHA; 3.0 mg/ml) for 2 days, followed by culture in medium containing interleukin-2 at 20 U/ml. Human recombinant interleukin-2 was obtained through the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health, from Maurice Gately, Hoffman-La Roche, Inc. [94]. Erythrocytes were collected from the bottom layer of the density gradients and washed twice with phosphate- buffered saline.
[00162] Neutrophils were isolated from healthy donors by Ficoll-Hypaque centhfugation. The bottom layer was collected, and erythrocytes were lysed by three treatments with ice-cold deionized water, followed by treatment with 2x Hanks balanced salt solution containing 5 mM HEPES buffer (Gibco BRL, Grand Island, N.Y.). To isolate platelets, blood was drawn in acid-citrate-phosphatedextrose anticoagulant (Biowhittaker) and centrifuged at 400 x g. The plasma fraction containing platelets was centrifuged (800 x g), washed twice with acidcitrate-phosphate-dextrose in 0.85% NaCI solution (1 :6, vol/vol), and resuspended in 0.85% NaCI until use, when the platelets were resuspended in complete medium [74].
[00163] Fresh tonsil tissues were obtained from the Pathology Department of Rush Medical Center. Tonsil mononuclear cells were isolated by teasing the tissues. Cells were washed and passed through a 70-mm nylon Spectra/Mesh filter (Spectrum Medical Industry, Inc., Houston, Tex.) to remove aggregated cells [74].
[00164] CD4+ T cells were positively isolated from fresh PBMC using anti-CD4 antibody-conjugated Dynabeads M-450 and CD4/CD8 DETACHaBEAD (Dynal, Oslo, Norway) in accordance with the manufacturer's protocols. After an additional positive selection of CD4+ T cells, the remaining cells were used as CD4- PBMC. The composition of the CD4+ and CD4- cell population as measured by flow cytometry, was .99% CD4+ and ,1 % CD4+ [74]. Virus stocks.
[00165] T-cell line-adapted (TCLA) HIV-1 strain MN (HIV-1 MN; AIDS Research and Reference Reagent Program [no. 317], contributed by Robert GaIIo) was grown in H9 cells. The 8E5/LAV cell line (AIDS Research and Reference Reagent Program [no. 95], contributed by Thomas Folks) was used to derive reverse transcriptase-defective virus and DNA for PCR standards [95]. The primary isolates of HIV (HIVGP [X4] and HIVTH [R5]) were produced in PHA-stimulated PBMC as previously described [96].
Binding of HIV to cells.
[00166] To measure binding of HIV to cells, a pellet of 5 3 106 erythrocytes or platelets or 1 3 106 Raji cells, neutrophils, total PBMC, CD4-depleted PBMC, purified CD4+ T cells, or tonsil mononuclear cells was incubated with 50 ml of virus containing approximately 1 ,000 pg of p24 for 2 h on ice. Cells were washed to remove unbound virus and transferred to fresh tubes since some virus binding to tubes occurred during incubation. Pelleted cells were treated with 0.5% Triton X-100, and the amount of virus bound to cells was detected by p24 antigen enzyme-linked immunosorbent assay (ELISA; National Institutes of Health AIDS Vaccine Program, Frederick, Md.). To protect neutrophil-bound HIV-1 p24 from proteolytic degradation, the protease inhibitors leupeptin (2 μg/ml), aprotinin (10 μg/ml), and phenylmethylsulfonyl fluoride (2 mM; Sigma) were added to the Triton X-100 solution. Tonsil-derived cells and PBMC were gamma irradiated with 5,000 rads [74]. [00167] Pelleted Raji cells were first fixed in a 0.5% formaldehyde solution for 10 min at room temperature. Cells were washed four times with serum-free medium, resuspended in complete medium, and then incubated with virus.
Incubation with HIV-1
[00168] In this method of assay, the HIV-1 virus is allowed to bind to a carrier in such a manner that the carrier-bound HIV-1 retains the ability to infect a target cell. Prior to such an infection the carrier-bound HIV-1 is exposed to candidate neutralizing antibodies. The unbound candidate neutralizing antibodies are then removed by washing the carrier free of unbound antibodies. As an example, if a cell lacking CD4 (a CD4-negative cell) were to serve as a carrier for binding to HIV-1 , and if the CD4- negative cell could not be infected by the HIV-1 , the HIV-1 would be retained on the surface of the cell where it could be tested for binding of antibody and the unbound antibody could be washed off by a variety of standard laboratory methods, such as simple centhfugation, or other methods of separating cells from the surrounding liquid milieu.
Infection of T cells by cell-bound and cell-free HIV.
[00169] Washed cells with bound virus (see above) or dilutions of cell-free virus were added to cultures of 2.5 x 105 H9 cells or PHA-stimulated PBMC in round-bottom polypropylene tubes (12 by 75 mm; Becton Dickinson, Franklin Lakes, N.J.). After 12 h, the cells were washed and cultured in 48-well cell culture plates (Corning Inc., Corning, N.Y.) for an additional 6 days and collected on day 7 [74]. P24 Antigen Assay
[00170] The total volume for all experiments was 500 ml. HIV-1 replication was assessed by measuring the p24 antigen in culture supernatants. In some experiments, to assess the effects of adhesion molecules on HIV replication, anti-LFA-1 β or anti- CD14 antibody was added at 1 μg/ml to H9 cells or PHA-stimulated PBMC 20 min prior to coculture with cell-bound virus. Antibody was maintained at 1 μg/ml throughout the coculture. Various P24 Antigen Assays, as known in the art and described herein, may be applied [74].
Examples of Post-Incubation Carrier Neutralization Assays
[00171] The resultant carrier-HIV complexes developed as, by way on nonlimiting example, disclosed in Examples 1-3 herein may be assayed by the various techniques described in the remaining examples once they have been properly incubated with the primary or clinical isolates of HIV-1 .
Example 4 PBMC Neutralization assay
[00172] Definitions and abbreviations
[00173] IMDM : Iscove's Modified Dulbecco's Medium
[00174] PBMC : Peripheral Blood Mononuclear Cells [00175] PBS : Phosphate Buffered Saline
[00176] TNC : Tri Sodium Citrate
[00177] PHA : Phytohaemagglutinine
[00178] FCS : Fetal Calf Serum
[00179] rlL2 : Recombinant interleukin-2
[00180] TCID50 : 50% Tissue culture infectious dose
Preparation of frozen, pooled PBMCs
[00181] PBMCs are isolated from buffycoats from 8 - 12 different HIV-negative healthy blood donors. PBMCs from each donor are isolated separately and are simultaneously tested for the presence of the CCR5 32bp deletion. PBMCs from donors homozygous for the 32bp deletion are excluded from the PBMCs pool.
[00182] Transfer the buffycoat to a 250 ml flask and take a 200 μl sample for the
CCR5 32bp deletion PCR. Add up the buffycoat to 200 ml with PBS supplemented with
10% TNC.
[00183] Add 12.5 ml Ficoll to each of eight 50 ml tubes and add 25 ml of diluted buffycoat on top of the ficoll in each tube.
[00184] Centrifuge the tubes for 20 min at 1000xg (acceleration 7, break 1 ) at RT.
[00185] Remove the ring fraction on top of the ficoll with a pasteur pipet and add 2 rings together in 1 clean 50 ml tube. Supplement the ring fractions with PBS/ 10% TNC to 50 ml.
[00186] Centrifuge the tubes for 15 at min 400xg (acceleration 9, break 7) at RT. [00187] Discard the supernatant and resuspend the cells in 50 ml PBS/ 10% TNC. [00188] Centrifuge the tubes for 10 min at 250xg (acceleration 9, break 9) at RT. [00189] Discard the supernatant and resuspend the cells in 50 ml PBS/ 10% TNC. Repeat the centhfugation and resuspension step until the supernatant is (almost) as clear as water.
[00190] When many erythrocytes are present, these can be removed by cell lysis. Resuspend the cells in 1 ml ACK lysing buffer before the last wash step, agitate gently for 1 minute, add up to 50 ml PBS/ 10% TNC, and centrifuge the tubes for 10 min at 250xg (acceleration 9, break 9) at RT.
[00191] PBMCs of different donors are pooled and frozen at a concentration of 50 million cells/ampoule (1.8 ml) in IMDM supplemented with 20% FCS and 10% DMSO.
PHA stimulation of PBMCs
[00192] Thaw the PBMCs in IMDM supplemented with 20% FCS and centrifuge the cells for 10 min at 400xg.
[00193] Resuspend the PBMCs in IMDM supplemented with 10% FCS, penicillin (100
U/ml), streptomycin (100 μg/ml), ciproxine (5 μg/ml) and PHA (200 ug/ml) to a concentration of 5 x 106 cells/ml.
[00194] Incubate at 370C, 10% CO2 for 3 days.
PBMC serum neutralization assay [00195] Neutralization assays are performed in triplicate in 96-wells plates, using 20 the carrier-virus complex and 105 PBMCs per well, in IMDM supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 μg/ml), ciproxine (5 μg/ml), rlL2 (20 units/ml), and polybrene (5 μg/ml). Two different protocols are in use, depending on the nature of the neutralizing agent (polyclonal or monoclonal). For polyclonal agents, smaller working volumes are used than for monoclonal agents. Moreover, a wash step is included in the polyclonal assay to reduce cytotoxic or other unwanted effects of the polyclonal reagent. In both protocols, virus production in culture supernatant at day 7 is analyzed by an in-house ELISA.
Polyclonal reagents
[00196] Add 25 μl medium to rows B till H.
[00197] Add 50 μl 2x neutralizing agent dilution to row A
[00198] Prepare serial dilutions of the neutralizing agents by pipetting 25 μl from row
A to row B, then mix, add 25 μl of row B to row C, mix, etc. up to row G from which 25 μl goes into waste.
[00199] Add 25 μl of virus dilution (800 carrier-virus complex/ml) to rows A till H.
[00200] Incubate for 1 hour at 370C, 10% CO2.
[00201] Centrifuge the 3-day PHA stimulated PBMCs for 10 min at 400xg, resuspend to concentration of 4 x 106 cells/ml and add 25 μl PBMC to rows A till H.
[00202] Incubate for 4 hour at 370C, 10% CO2. [00203] Centrifuge the PBMCs for 3 min at 400xg (acceleration 9, break 4), remove the medium and add 150μl PBS. Centrifuge the PBMCs 3 min 40Og (acceleration 9 break 4), remove the PBS and add 150μl medium.
[00204] Incubate at 370C, 10% CO2.
[00205] Analyze p24 production in culture supernatant at day 7 by ELISA.
Monoclonal reagents
[00206] Add 50 μl medium to rows B till H.
[00207] Add 100 μl 2x neutralizing agent dilution to row A
[00208] Prepare serial dilutions of the neutralizing agents by pipetting 50 μl from row
A to row B, then mix, add 50 μl of row B to row C, mix, etc. up to row G from which 50 μl goes into waste.
[00209] Add 50 μl of virus dilution (400 carrier-virus complex/ml) to rows A till H.
[00210] Incubate for 1 hour at 370C, 10% CO2.
[00211] Centrifuge the 3-day PHA stimulated PBMCs for 10 min at 400xg, resuspend to a concentration of 2 x 106 cells/ml and add 50 μl PBMC to rows A till H.
[00212] Incubate at 370C, 10% CO2.
[00213] Analyze p24 production in culture supernatant at day 7 by ELISA. Example 5 PBMC Neutralization Assay with RT-PCR
Materials
[00214] RPMI 1640 powder 10 packs
[00215] Fetal bovine serum 10 bottles
[00216] P24 ELISA kit (Organon Teknika) 10 packs
[00217] QIAamp viral RNA mini kit (250 tests) 1 pack
[00218] HIV RG RT PCR (Artus) (24 tests) 10 packs
[00219] IL-2, PHA, Penicillin, Streptomycin
[00220] 96 well microtiter plate
[00221] LightCycler Capillaries 3 packs
[00222] 15 ml test tubes
Methods
Preparation of donor PBMCs
[00223] The buffy coat is diluted with equal volume of PBS and then 30 ml of cell suspension is gently placed on the top of 15 ml Ficoll-hypaque in a 50 ml centrifuge tube. Centrifuge at 1 ,500 rpm for 20 min. The PBMCs at interface are collected and transferred to new tube and wash with RPMI 1640. After that, the PBMCs are added with PHA medium for adjust to 1x106 cells/ml. The PBMCs are maintained in tissue culture flask at 37 0C, 5% CO2. After 2-3 days, the PHA medium is removed and replaced with IL-2 medium for 1 day before adding to HIV-1 co-culture or TCID50 testing or neutralization test.
HIV-1 carrier complex propagation and co-cultivation
[00224] Carrier complex viral stock is prepared by infection of PHA stimulated PBMCs with 200 μl of HIV-1 positive supernatant. After overnight incubation, the infected PBMCs are washed with RPMI 1640. Then 4 ml of IL-2 medium are added and cultured in tissue culture flask at 370C, 5% CO2. The p24 antigen is detected by ELISA kit (Organon Teknika) every week. The culture medium is harvested as soon as the p24 positive in high titer. Then TCID50 is determined and stored the supernatant at -80 0C for neutralization test.
Tissue culture infectious dose (TCID50)
[00225] 200 μl of supernatant from HIV-1 co-culture is diluted by 5 fold dilution. The dilution is performed in 6 dilutions per one virus. Then 100 μl of each dilution are incubated with 100 μl of PHA stimulated PBMCs in 15 ml test tube at 37 0C, 5% CO2 for overnight. Then wash with RPMI 1640 for 3 times and add 1 ml of IL-2 medium to each tube. After that, 200 μl of cell suspension are transferred to each 5 wells of 96-well microtiter plate and incubated at 37 oC, 5% CO2. On day 4, the medium is changed and day 8, the culture medium is collected to measure p24 antigen. TCID50 is calculated according to the Spearman-Karber formula (see Validation of Pharmaceutical Processes By James P. Agalloco, Frederick J. Carleton Edition: 3, illustrated Published by CRC Press, 2007, pp 170-171.)
Neutralization test (Infectivity reduction assay calculated by RNA quantitation measured by real time RT-PCR)
[00226] This method estimates neutralization of HIV-1 positive serum or plasma by fixing antibodies in serial dilutions of viral carrier complex. Then the viral load of each dilution is obtained at day 2 by using real time RT-PCR kit (Artus). The ratio of TCID50 of seronegative serum and test serum is calculated and reported.
[00227] This method for detection one virus and one antibody
[00228] 1. Heat inactivated serum for 30 min at 56 oC.
[00229] 2. Dilute seronegative serum and test serum to 1 :30, or antibody with IL-2 medium.
[00230] 3. Dilute virus HIV-1 carrier complex from culture to 100 TCID50 with IL-2 medium for 1 ,400 μl. Then dilute virus HIV-1 carrier complex by serial 2 fold dilution from 100 to 6.25 TCID50.
[00231] 4. Incubate 75 μl of various virus HIV-1 carrier compolex dilutions with 75 μl of seronegative serum or positive control serum or test serum/antibody in test tubes at
37 oC, 5% CO2 for 1 h. [00232] 5. Prepare 1 ml of 1.34x106 cell/ml PHA activated PBMC in IL-2 medium.
[00233] 6. Add 75 μl of prepared PBMC in all test tubes and incubate at 37 oC, 5%
CO2 for overnight.
[00234] 7. Wash 3 times with 10 ml of RPMI-1640.
[00235] 8. Centrifuge and remove supernatant.
[00236] 9. Add 400 μl of IL-2 medium in all test tubes and place 200 μl of suspension into 2 plates of 96-well plate. One is incubated at 37oC, 5% CO2 for 2 days. The other plate is incubated for 8 days and change medium at day 4.
[00237] 10. The supernatants from the first plate (2 days) are collected to RNA extraction by using QIAamp viral RNA mini kit (QIAGEN). Then 5 μl of RNA are used to determine viral load by real time RT-PCR. The real time RT-PCR is performed by using
LightCycler (Roche) and using HIV 1 RG RT PCR (Artus) as reagent.
[00238] 11. For the other plate (8 days), 100 μl of supernatants are collected and performed by using p24 ELISA test kit (Organon Teknika).
[00239] 12. The values of viral load and p24 are collected and analyzed. The neutralizing index is expressed as a ratio of the TCID50 obtained in the presence of
HIV-1 seronegative over that obtained in the presence of test serum. The neutralizing index < 3 is considered negative, while neutralizing index 3-9, 10-100 or >100 are considered as weak, medium and strong neutralizing activity, respectively.
Viral load detection by real time RT-PCR [00240] 140 μl of supernatant from neutralization test are extracted by QIAamp viral RNA mini kit (QIAGEN) and RNA is eluted by 60 μl of elution buffer. This RNA is collected and kept at -80 oC until use. 8 μl of RNA are used to determine viral load.12 μl of master mix in HIV 1 RG RT PCR kit (Artus) and 8 μl of RNA are added in LightCycler capillary tube. Then RT-PCR is performed and using Taqman probe which specific for HIV-1 for detect viral load at channel F1/F2.
Reagents
[00241] RPMI 1640 medium - Dissolve 1 pack of RPMI 1640 powder in 950 ml of deionized distilled water by stirring. Add 2 g NaCO3 and adjust pH to 7.2 with 1 N HCI. Add deionized distilled water to 1 ,000 ml. Sterilize by filtering through 0.45 μm millipore filter. Store at 4 0C until use.
[00242] IL-2 medium - Mix 90 ml of RPMI 1640 medium, 10 ml of Fetal bovine serum, 100 μl of IL-2 (10,000 U/ml), 1 ml of L-glutamine, 200 μl of penicillin (50,000U/ml) and 200 μl of Streptomycin (50,000 U/ml).
[00243] PHA medium - Mix 90 ml of RPMI 1640 medium, 10 ml of Fetal bovine serum, 20 μl of PHA (2.5 mg/ml), 1 ml of L-glutamine, 200 μl of penicillin (50,000U/ml) and 200 μl of Streptomycin (50,000 U/ml). EXAMPLE 6 PBMC Neutralization assay using HIV-1 p24 Antigen
[00244] In this microtiter plate neutralization assay, virus infection in donor PHA- stimulated PBMC is assessed by a quantitative ELISA measurement of HIV-1 p24 antigen expressed in PBMC culture supernatants.
Materials and Equipment
[00245] Biological safety cabinet class Il
[00246] Humidified 37°C incubator with 5% CO2.
[00247] Centrifuge equipped with microplate carriers
[00248] Electric Pipettor
[00249] Adjustable pipetters
[00250] Multichannel pipetters
[00251] Twelve-channel manual ELISA aspirator for washing cells in PGC microtiter box (Drummond Scientific)
[00252] Vmax ELISA plate reader with Soft Max Pro software
[00253] 96 well plate washer
[00254] Vacuum trap suction
[00255] Microscope
[00256] Hemocytometer
[00257] Disposable plastic pipettes [00258] 1 -250μl pipette tips
[00259] 50ml conical centrifuge tube
[00260] 96-well flat bottom plates
[00261] 96-well round bottom plates
[00262] 96-well microtiter (yellow) boxes
[00263] Plate covers
[00264] Micotiter plate sealers
[00265] Sterile reagent reservoirs
[00266] IL-2 media
[00267] Wash media
[00268] PBS-Dulbecco's Phosphate-buffered saline without calcium and magnesium
[00269] PHA-P prepared in sterile distilled water to 1 mg/ml.
[00270] Target cells: Ficoll gradient separated PBMC from an H IV-1 -seronegative donor.
[00271] H IV-1 p24 antigen ELISA kit (Advanced Bioscience Laboratories, ABL)
[00272] Pooled normal human serum (NHS)
[00273] Control pool of serum from HIV-1 positive individuals
[00274] Trypan blue: 0.4 %
[00275] 70 % ethanol
Definitions and Abbreviations
[00276] PPE Personal Protective Equipment [00277] PBS Phosphate Buffered Saline
[00278] BSC Biological Safety Cabinet
[00279] PBMC Peripheral Blood Mononuclear Cell
Procedure
[00280] Day 0
[00281] Count the PHA-stimulated PBMC, those stimulated three to four days prior to the assay. The viability should be greater than 90%. Resuspend cells in IL-2 medium at 3x106 cells/ml.
[00282] Prior to use, ensure that all test sera and control sera have been complement depleted by heat inactivation for 45 minutes at 56°C. Purified antibodies or peptide reagents do not need to be heat inactivated.
[00283] Dilute sera in a 96-well flat bottom plate (<200ul/well) or titer tube box (>200u I/well) using IL-2 medium as a diluent. Calculate amounts for number of operators and triplicate wells with 20 % extra. Aliquot diluted serum into the deep-well plate (25 μl/well). To avoid air bubbles in the wells, when aliquoting place the tips of the multichannel pipette at the bottom of wells. [00284] Set up one row (at least 6 wells) with IL-2 medium only, this will serve as a baseline for virus growth (it is referred to as the virus only or regular medium row).
[00285] Thaw quickly in a 37°C water bath one aliquot of virus carrier complex stock. Dilute it with IL-2 medium to the concentration calculated from the viral titration. Add 25μl of diluted viral stock to the side of each well of the deep-well plate. Be careful not to touch the tips to the plate or the serum at the bottom of the wells. After dispensing virus carrier complex gently tap the deep-well plate to ensure that serum and viral complex are well mixed in the bottom of the wells. Incubate plates at 37°C for thirty minutes.
[00286] Add 50μl of PHA-stimulated PBMC, resuspended at 3x106 cells/ml, to each well, gently tap the plate to mix PBMC with sera/virus. Cover the plate with loose fitting plate lid and incubate overnight (at least 18 hours) at 37°C, 5%CO2.
[00287] Day 1
[00288] The next day, wash the deep well plates three times by filling wells to 500μl with Wash medium (first 2 washes) and IL-2 medium (last wash). Seal plate with adherent plastic plate sealer and centrifuge for 10 minutes at 1200 rpm.
[00289] After each wash aspirate supernatants from each well with an aspirator set to leave 50μl well. Bring the volume up to 500μl in each well and then centrifuge again. Repeat with IL-2. [00290] After the last wash add 200ml of IL-2 medium to each well. Thoroughly resuspend cell pellets and transfer 220ml from the deep-well plate to a round bottom 96-well microplate. Inspect plates daily for uniformity in size of cell pellets and pH of culture media. Incubate at 37°C, 5%CO2 for 3 days.
[00291] Day 4/Day6
[00292] Measure p24 antigen production on days 4 or 6. Remove 50μl of culture supernatant from each well and place into a corresponding 96-well flat bottom plate with 15OuI of 1 :4 Disruption buffer (provided in the ABL ELISA kit), diluted in PBS (final overall dilution 1 :4).
[00293] Using ABL p24 ELISA kit, determine p24 concentration in Virus Only row of the experiment plate If the concentration is > 10 ng/ml four days after infection, analyze corresponding sera samples. The harvest p24 plate can be stored at -200C until p24 determination is complete. If the experiment needs to be continued, <10 ng/ml p24, add 100ml of fresh IL-2 media to each well of the plate and incubate at 37°C.
[00294] Repeat collection of culture supernatants six days after infection and check the p24 concentration.
[00295] Calculate percent neutralization and IC50,80, 90. Record data. Examples of Quantification Protocols after Competitive Binding in Carrier Neutralization Assays
[00296] The resultant carrier-HIV-neutralizing antibody complexes developed as, by way on nonlimiting example, disclosed in Examples 1-6 herein may be alternately assayed by the various techniques described in the remaining examples.
Example 7 Anti-HIV-1-gp120env Antigen Elisa Protocol
Introduction:
[00297] This assay can be used to detect HIV-1 gp120 proteins or anti-gp120 antibodies. In brief, an antibody (D7324) adsorbed on a microtiter plate captures gp120 from solution. The captured gp120 protein is then recognized at a separate site by a second antibody that is, in turn, detected using a third, enzyme labeled, anti-antibody. In its original version, the sandwich ELISA utilized an alkaline-phosphatase (AP) label and the AMPAK ELISA amplification system in the color-development stage1 ,2,3. However, other enzyme labels (horseradish peroxidase, luciferase) and the appropriate detection systems can often be substituted for the AP system.
Materials: [00298] Capture antibody Product code D7324, supplied by Aalto Bio Reagents, Dublin, Ireland, is produced by first immunizing sheep with a single synthetic peptide which has the amino acid sequence : APTKAKRRVVQREKR
[00299] This amino acid sequence corresponds to amino acid numbers 497-511 in the envelope gene gp120 protein of the BH-10 strain of HIV-1. The antibodies are isolated from the sheep hyperimmune serum by affinity chromatography using the above synthetic peptide coupled to Sepharose.
Detection antibody:
[00300] For detection and quantification of gp120, any specific anti-gp120 MAb can be used provided that it is able to recognize the particular gp120 protein under evaluation and has a high enough affinity. The bound MAb is then detected using an enzyme-labeled anti-antibody of appropriate specificity. This protocols covers the use of AP- label anti-antibody.
[00301] For detection of antibodies to gp120 (e.g., in the sera of vaccinated animals or HIV-infected humans), the antibody preparation is titrated then the bound antibodies are detected as described above.
HIV-1 gp120 Standard : [00302] Recombinant HIV-1 gp120 Antigen, product code BR 6106, supplied by Aalto Bio Reagents, Dublin, Ireland.
Methods :
[00303] 1. D7324 is reconstituted in distilled water at 1 mg/ml and stored in frozen aliquots.
[00304] 2. D7324 is coated onto lmmulon Il microelisa plates (Dynatech Ltd.) by incubating them for 12-18 hours at room temperature in 10Oμl per well of 10OmM NaHCO3,. The optimal antibody concentration is 5μg /ml.
[00305] 3. The wells are washed twice with 200μl of Tris-Buffered Saline (TBS; 144mM NaCI, 25mM Tris, pH 7.6), blocked for 30 minutes with 200μl of a solution of 2% non-fat milk powder (Marvel, Cadbury Ltd.) in TBS, and then washed again with TBS.
[00306] 4. HIV-carrier complex sample is added to the wells in 10Oμl of TBS (a detergent such as 1 % NP40 can be added but it is usually not necessary) and incubated for 2 hours at room temperature.
[00307] 5. Unbound protein is removed washing twice with TBS (200μl) and captured gp120 is detected by addition for 1 hour of a second antibody, as outlined above. The antibody is diluted in TMT/SS buffer (4% nonfat milk powder and 0.5% Tween-20 in TBS plus 20% sheep serum, 10Oμl per well). The concentration of detection antibody to be used must be determined empirically, e.g., by titration.
[00308] 6. Unbound antibody is removed by washing the wells three times with TBS (200μl), then the AP- labelled detection antibody is added for 1 hour in TMT/SS buffer.
[00309] 7. Unbound antibody -AP is removed by washing the wells six times with 200μl of AMPAK wash buffer, and bound antibody -AP is detected with the AMPAK-II ELISA amplification system (Dako Diagnostics) essentially as recommended by the manufacturer 2,3. The reactions are stopped with 50μl of 0.5 M HCI and the absorbance determined at 492nm. Alternative detection systems may also be suitable, as noted above.
[00310] 8. The HIV-1 gp120 assay is calibrated using known amounts of purified recombinant HIV-1 gp120 Antigen (product code BR 6106). It is recommended to assign a formal HIV-1 gp120 concentration for product code BR 6106 with reference to a commercial assay for which formally assigned standards are available.
Example 8 HIV-1 p24 Gag Antigen Elisa - Chemiluminescent Substrate
Introduction [00311] The assay is a twin-site sandwich ELISA. Briefly, p24 antigen is captured from a detergent lysate of virions by a polyclonal antibody adsorbed to a solid phase. Bound p24 is detected with an alkaline phosphatase-conjugated anti-p24 monoclonal antibody and a luminescent detection system. The luminescence readout gives a broader dynamic range (2.5 logs versus 1 log with the AMPAK system) and an extended linear range which allows more accurate quantification. Furthermore, the broader dynamic range in this system makes the testing of serial dilutions of samples unnecessary in most cases. It also makes it unnecessary in most cases to re-analyze samples because their p24 content is out of linear range. This reduces the overall number of ELISA samples to be tested and minimizes handling and sample preparation times.
Antibodies
[00312] 'Coating antibody D7320, sheep anti-HIV-1 - p24 gag, affinity purified, 2mg/vial; store at +2 to 8oC. This is supplied by Aalto Bio Reagents Ltd., Dublin, Ireland.
[00313] This is a mixture of 3 sheep polyclonal antibodies raised against peptides from the HIV-1 (LAV-1 ) sequence, then affinity purified with the respective immunogenic peptides. The amino acid sequences used are :
[00314] SALSEGATPQDLNTML aa 173 - 188 [00315] GQMREPRGSDIA aa 226 - 237 [00316] LDIRQGPKEPFRDYV aa 283 - 297
[00317] These sequences are substantially conserved between HIV-1 isolates.
[00318] Secondary conjugate antibody code BC 1071 -AP, alkaline phosphatase conjugate of anti-HIV-1 -p24 mouse monoclonal, 50μl/vial; store at +2 to +8 0C. This is supplied by Aalto Bio Reagents Ltd., Dublin, Ireland. This antibody is an alkaline phosphatase conjugate of monoclonal clone EH12E1. EH12E1 is a mouse monoclonal antibody raised against HIV-1 (CBL-1 ) and mapped to a complex epitope incorporating two distinct peptide sequences as follows :
[00319] GHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQ (aa 193-227); and, [00320] NPPIPVGEIYKRWII (aa 253-267).
[00321] These regions of p24 are conserved between HIV-1 strains, and also substantially between HIV-1 and HIV-2.
Reagents, Buffers
[00322] TROPIX ELISA-Light Immunoassay system: EL100CX chemiluminescent substrate for alkaline phosphatase with enhancer (CSPD with Sapphire-ll)
[00323] 10 x NaHCO3: 42g. NaHCO3 (SIGMA Cat No S-6014) add 500 ml dd H20, pH=8.5 (keep in fridge) [00324] 1 x TBS 144mM NaCI; 25mM TRIS, pH 7.5
[00325] 10 x TBS for 5 liters: 421 g. NaCI (SIGMA Cat No S-9888); 151g. Tris-Trizma base (SIGMA Cat No T-1503); dd H20 add to 5 liters; pH 7.5 (use HCI to adjust)
[00326] 10 x PBS for 10 liters: 80Og. NaCI; 20 g. KCI; 144 g. Na2HPO4; 24 g. KH2PO4; fill up to 8 liters with H20; adjust to pH 7.4 with HCI; fill to 10 liters.
[00327] 10 x TROPIX wash buffer For 1 liter: component of ELISA-Light Kit Cat No EL100CX 100 ml 10 x assay buffer concentrate 900ml ddH20
[00328] 0.1 % Empigen in TBS: Empigen BB Detergent -35% solution, SIGMA Cat No 45165
[00329] 0.1 % Tween in PBS: Tween-20 ,SIGMA Cat No T-1379
[00330] p24 Standard: Recombinant HIV-1 p24 from Aalto Bio Reagents, Code AG 6054; Dilute with 1 % FCS in TBS to 100μg/ml; Store in 100μl aliquots at -20 0C.
[00331] Sheep serum: SIGMA Cat No S-7773
[00332] 2% milk (for 40 plates) : blocking buffer; 10g. non-fat dry milk (Carnation); 500ml 1 X TBS; Stir for one hour and filter twice through folded felt; Paper Reeve Angel (24.0 cm) [00333] Conjugate: Dilute BC 1071 -AP in 20% sheep serum, 0.05% Tween, 1 x TBS
[00334] Plates: COSTAR white opaque 96-well plates, high binding (Cat No 3922) Assay Coating p24 Plates.
[00335] Day 1 : Re-suspend antibody D7320 in 1 ml dd H2O (2mg/ml)
[00336] Add the 1 ml solution of D7320 to 400ml of 0.1 M NaHCO3 , pH = 8.5 (40ml of 10 x NaHCO3 + 359ml dd H2O), thereby adjusting the antibody concentration to 5 μg/ml.
[00337] Stir 2 - 3 minutes
[00338] Add 100μl/well, then, stack the plates. Place a cover plate on top and wrap with saran wrap to avoid evaporation and incubate overnight at room temperature.
[00339] Note : one 2mg vial of D7320 is sufficient to coat 40 x 96-well plates. In contrast to the previous "AMPAK" protocol, all wells of the 96-well plate can be used for assaying. There is no difference in coating and readout between outside and central wells.
[00340] Day 2 Prepare 500ml 2% milk in 1 x TBS: Stir for one hour at room temperature; Filter twice with Reeve Angel filter paper. [00341] Wash plates twice with 1 x TBS (200ml buffer per well). Add 100ml/well of 2% milk, then stack plates, place a cover plate on top and with saran wrap to avoid evaporation and incubate for one hour at room temp.
[00342] Freeze at -20 0C. (Coated plates can be stored for several weeks).
p24 ELISA Assay
[00343] Thaw coated p24 plates.
[00344] Discard buffer in wells and wash once with 1 x TBS.
[00345] Discard TBS and tap plates dry.
[00346] Prepare p24 standard as needed, dilute standard in 0.1 % Empigen in TBS
[00347] HIV-1 -Carrier-Neutralizing Antibody Complex Samples to be analyzed should be treated with 1 % Empigen prior to assaying to inactivate the virus and linearize the proteins. (Note: The addition of the detergent is necessary. Omitting the detergent treatment or using other detergents like Tween-20 and NP40 seriously inhibits capture by D7320).
[00348] Add 10Oμl of samples, standards and controls to the appropriate wells and incubate for 3 hours at room temperature. To avoid evaporation, blank wells are filled with 0.1 % Empigen in TBS and plates are sealed with saran wrap.
[00349] Plates are washed twice with 1x TBS, then tapped dry. [00350] 10Oμl of conjugate is added per well (Note: each lot of the secondary antibody conjugate preparation has to be individually tested for the optimal concentration to be used. The optimal dilution range of the product usually lies between 1 :5000 - 1 :10000.
[00351] Incubate for one hour at room temperature, then, wash 4 times with 0.1 % Tween in PBS
[00352] Wash plates 2 times with TROPIX wash buffer and tap dry.
[00353] Add 50 μl/well of CSPD with Sapphire Substrate (TROPIX)
[00354] Incubate for 30 minutes at room temperature.
[00355] Read plates in luminometer
Example 9 Anti-HIV-1 -gp120gag Antigen Elisa Protocol - Biotinylated Conjugate
Buffers
[00356] Coating buffer 10OmM NaHCO 3 pH 8.5; 8.4g in 1 L = pH 8.0; «450μl 10M NaOH in 1 L gives pH 8.5 [00357] 1 OxTBS 1.44M NaCI, 0.5% Tween 20, 25OmM Tris pH 7.5; 168.32g NaCI in 2L; 10ml neat Tween 20 in 2L; 60.6g Tris in 2L gives pH 10.1 ; « 30ml Cone. HCL in 2L gives pH 7.5
[00358] Blocking buffer 0.4g milk powder in 20ml IxTBS 2g milk powder in 100ml enough for 4-5 plates.
[00359] PBS / E / S PBS with 0.1 % empigen and 10% lamb serum 9ml PBS, 1 ml serum, 70μl empigen for 10ml
[00360] TMT / SS IxTBS with 2% milk, 20% lamb serum, 0.05% Tween 20
TABLE A
25ml 50ml 100ml 150ml 200ml
Water (ml) 16.25 32.5 65 97.5 130
1OxTBS (ml) 2.5 5 10 15 20
Milk (g) 0.5 1 2 3 4
Serum (ml) 5 10 20 30 40
Tween 20 (ml; 10%) 1.25 2.5 5 7.5 10
[00361] Reaction buffer 1OmM ethanolamine, 0.5mM MgCI2, pH 9.8; 0.61 ml ethanolamine in 1 L; 0.5ml MgCI2 (1 M stock) in 1 L gives pH 10.2; «160μl Cone. HCI gives pH 9.8 TABLE B
Item Catalog No. Company Units
Coating anti-H I V-1-p24 D7320 Aalto 2mg (2ml)
Biotin anti-HIV-1-p24* BC 1071 -BIOT* Aalto 100μl
Streptavidin-AP 1089 161 Boehringer 1 ml, 1000U pNPP substrate S0942 Sigma 5mg tablets
[00362] A suitable method of medium-term storage after receipt is to dilute 0.1 ml of the biotinylated conjugate to 1.0ml (1 :10) with TMT / SS buffer. Then aliquot and store 0.1 ml volumes (sufficient for 1 plate) at -200C. Prior to use, dilute 0.1 ml to 10ml (1 :100) in TMT / SS buffer.
Protocol
[00363] Coating the plate: Add 100μl of D7320 (diluted to 1 mg/ml) to 9.9ml coating buffer and add 10Oμl to each well. Cover and leave overnight at room temperature.
[00364] Wash with 1 xTBS for 3x200μl
[00365] Add 200μl of blocking buffer to each well for 30 minutes
[00366] Wash with 1 xTBS for 3x200μl [00367] Place 10Oμl of HIV-Carrier-Neutralizing-Antibody complex supernatant (previously treated with 0.1 % - 0.2% empigen and heat inactivated for 30 minutes at 56°C) into well. For ELISA controls either add duplicate 100ng/ml p24 Ag standards (1 :10 dilution of aliquot in PBS / E / S) or use a standard curve; 46.25μl transfer volume in 100μl (46.25 / 146.25 = 316) to give 1000, 316 100, 31.6 10, 3.16 and 1 ng/ml. Use PBS / E / S as negative controls. Cover and leave overnight at room temperature.
[00368] Wash with 1 xTBS for 6x200μl
[00369] Dilute 0.1 ml of stock solution of biotinylated antibody conjugate to 10ml (1 :100) in TMT / SS. Then add 10Oμl per well and leave at room temperature for 2 hours.
[00370] Wash with 1 xTBS for 6x200μl
[00371] Dilute 20μl of streptavidin-AP reagent in 10ml TMT / SS. Add 100μl to each well and leave at room temperature for 1 hour.
[00372] Wash with 1 xTBS for 6x200μl
[00373] Add 100μl (2x5mg tablets in 10ml reaction buffer) pNPP solution to each well. Place 10Oμl of same in first column off a blanking plate. Incubate in the dark until the O. D. of 100ng/ml control >1.000. Read at 405nm wavelength. Example 10 HIV Neutralization with TZMBL Cells
Materials
[00375] A. Biological safety cabinet, class Il
[00376] B. Incubator, 37° C, 5% CO2
[00377] C. Centrifuge equipped with microplate carriers
[00378] D. Compound microscope
[00379] E. Inverted microscope
[00380] F. Hemocytomer
[00381] G. Luminometer (Perkin Elmer)
[00382] H. PipetteAid & Stripette tips (5ml, 10ml, and 25ml)
[00383] I. Single and multichannel pipetters & pipette tips
[00384] J. T-75 culture flask
[00385] K. Black-bottom, flat-bottom 96-well microplate (Culture Plate 96-F) (Perkin
Elmer)
[00386] L. Clear-bottom, flat-bottom 96-well microplate (View plates) (Perkin Elmer)
[00387] M. 50 ml conical tubes
[00388] N. Dulbecco's Phosphate Buffer Saline, (PBS)
[00389] O. Complete DMEM containing 15% heat-inactivated FBS, L-glutamine, and penicillinstreptomycin
[00390] P. Trypsin (Quality Biological) [00391] Q. 37°C Water Bath (180 Series Precision Scientific, Winchester, VA)
[00392] R. DEAE-Dextran
[00393] S. Cultured TZM-bl cells.
[00394] T. Luciferase Britelite Reconstitution Buffer (PerkinElmer)
[00395] U. Luciferase Britelite Substrate (PerkinElmer)
[00396] V. Pseudovirus-Carher Complex stock
[00397] W. Test neutralizing reagents
Definitions and Abbreviations
[00398] A. TZM-bl = a luciferase expressing reporter cell that possesses CD4 and both CXCR4 and CCR5 coreceptors
[00399] B. DMEM = DulbeccoA/ogt Modified Eagle's Minimal Essential Medium
[00400] C. cDMEM = complete DMEM media
[00401] D. FBS = Fetal Bovine Serum
[00402] E. RLU= Relative Light Unit
Procedure
[00403] All incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.
TZM-bl Neutralization Assay [00404] 1. Dilute test neutralizing reagents to desired concentrations.
[00405] 2. Add 25μl_ of test plasma/sera/antibodies to the microplates. The first microplate for each assay should be a black, clear-bottom view plate. Additional microplates can be the black-bottom culture plate.
[00406] 3. Dilute test pseudovirus-carrier complex sample to desired concentrations.
[00407] 4. Add 25μl_ of pseudovirus-carrier complex sampole to the microplates.
[00408] 5. Incubate all microplates at 37oC, 5% CO2 for thirty minutes.
[00409] 6. While the microplates are incubating, count and resuspend the TZM-bl cells in cDMEM. Trypsinize a flask of TZM-bl cells 20 minutes prior to use. Remove all the media from a flask of grown TZM-bl cells with a sthpette. Add 5ml of PBS to the flask and lightly swirl for up to one minute. Remove all PBS with a stripette. Add 3mls of
Trypsin to the flask and incubate for five minutes at 37 0C, 5% CO2. Transfer this 3mls of cells to a 50ml conical tube containing 27ml cDMEM. Centrifuge for ten minutes at
1200rpm. Resuspend these cells at 2x105 cells/mL in cDMEM containing a concentration of 60ug/ml DEAE-dextran, final concentration 30ug/ml.
[00410] 7. When the thirty minute incubation is complete, add 50μl_/well of the resuspended TZM-bl cells to the microplates. Return the microplates to incubate at 37
0C, 5% CO2 for 48 hours.
Analysis of TZM-bl neutralization [00411] 1. After a 48 hour incubation period prepare the Britelite substrate for plate analysis. Reconstitute the lyophilized luciferase Britelite substrate by adding luciferase
Britelite reconstitution buffer, 1 OmIs buffer into a 10ml substrate bottle. Mix and keep at room temperature.
[00412] 2. Before adding substrate to the microplates, observe the clear-bottom view plate under an inverted microscope for confluence.
[00413] 3. Add 10Oμl Britelite substrate to all wells of the microplate. Wait one minute before removing the microplates from the hood.
[00414] 4. Read plates using a Victor luminometer, use the Luminescence plate protocol.
[00415] 5. Average the RLU value for each dilution of neutralizing reagent and compute the IC50, 80 and 90 values. Record along with percent neutralization.
Example 11 Results of Carrier Neutralization Assay
[00416] The assay utilized erythrocytes, separated from a leukopack, as virus carrier. The erythrocytes were washed 3 times in PBS, stored in Adsol solution at 4oC until use. Two milliliters of the erythrocyte suspension (in 5x109/ml density) in RPMI medium and 2 ml of the HIV-1 stock (BZ167) were mixed together in a 14 ml polypropylene tube, incubated at 40C for two hours, then washed three times in PBS each followed by a centhfugation at 1500 rpm at 40C. After the last wash and centrifugation the erythrocyte-bound viruses were resuspended in RPMI medium, and 100 μl of the suspension was transferred to a polypropylene deep-96-well plate. Twenty microliters of the human monoclonal IgG mAb, b12, was added to each corresponding well in fourfold dilutions (0.06 - 64 μg/ml). The carrier-virus was incubated with the mAb at 370C for 1 hour, then washed in IL-2 RPMI, and 100 μl of the erythrocyte-virus-antibody solution was transferred to a polypropylene cell culture plate. Fifty microliters of PBMC as target cell were added to each well. When the target cells were mixed with carrier- bound virus, the virus transferred to the target cells, thereafter causing infection in trans. On day 4, postinfection, the p24 as a hallmark of the amount of the progeny viruses were measured by the standard p24 capture ELISA kit (ABL Inc.). The manufacturer's protocol was followed with a slight modification. Briefly, viral particles in the culture supernatant were disrupted, inactivating the virus and releasing the p24 into solution to enable detection. The microtiter wells were coated with two murine monoclonal antibodies that react with HIV-1 p24. Test samples were added to the wells, and unbound materials were then thoroughly washed away. The conjugate containing peroxidase-conjugated human anti-p24 polyclonal antibodies were added. After washing away the unbound conjugate, the peroxidase substrate was added. The color intensity were quantified by reading the absorbance at 450 nm. The concentrations of p24 in the samples were interpolated from a p24 standard curve.
[00417] The results of this assay are shown in FIG. 8.
[00418] Various features of novelty that characterize the invention are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and specific objects attained by its uses, reference is made to the accompanying drawings and descriptive in which a preferred embodiment of the invention is illustrated.
[00419] Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.
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Claims

CLAIMSWhat is claimed is:
1. A method of performing an assay comprising the steps of: incubating a carrier with a test sample to produce a first complex; incubating an analyte with said first complex to produce a second complex; and, performing a measurement on said second complex.
2. The method of claim 1 , wherein the carrier is a CD4- cell.
3. The method of claim 2, wherein the CD4- cell is selected from a group consisting of Raji cells (B lymphocytes), erythrocytes, platelets, and neutrophiles.
4. The method of claim 2, wherein the carrier is a subunit of a CD4- cell.
5. The method of claim 1 , wherein the test sample is selected from a group consisting of human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; and biological fluids such as cell culture supernatants; fixed tissue specimens; and fixed cell specimens.
6. The method of claim 1 , wherein the test sample is an enveloped virus.
7. The method of claim 6, wherein the enveloped virus is HIV.
8. The method of claim 1 , wherein the measurement is a neutralization assay.
9. The method of claim 8, wherein the neutralization assay is selected from a group consisting of assays as described in Table A herein.
10. The method of claim 8, wherein the neutralization assay is selected from a group consisting of assays as described in Fig. 3 herein.
11. The method of claim 1 , wherein the first complex carrier- retains the ability to infect a test sample without infecting said carrier;
12. The method of claim 1 , wherein the analyte is a neutralizing antibody;
13. An assay, wherein said assay allows direct interaction of potentially neutralizing antibodies with HIV-1 itself in the absence of host cell (or host carrier/particle) or target cell interactions.
14. The assay of claim 13, wherein said assay allows subsequent measurement of the ability of the HIV-1 that has been exposed to said potentially neutralizing antibody, for its ability or inability to infect a target cell.
15. A method of assay, comprising of the steps of incubating HIV-1 with a carrier particle or carrier cell in such a manner that the carrier-bound HIV-1 retains the ability to infect a target cell without infecting said carrier particle or carrier cell; exposing the carrier-bound HIV-1 to candidate neutralizing antibodies; removing unbound candidate neutralizing antibodies; and, measuring the level of neutralization of trans-infection of the carrier-bound the HIV-1.
16. The method of claim 15, wherein the unbound candidate neutralizing antibody could be washed off by a variety of standard laboratory methods, such as simple centrifugation, or other methods of separating cells from the surrounding liquid milieu.
17. A neutralization assay comprising a CD4- carrier complexed with HIV-1 , pretreated with a neutralizing antibody and evaluated for percent neutralization of trans-infection.
18. The neutralization assay of claim 17, wherein said CD4- carrier comprises erythrocytes.
19. The neutralization assay of claim 18, wherein the erthyrocytes are examined for the effects of antibody-mediated complement activation by lysis assay.
20. The neutralization assay of claim 17, wherein said assay is enhanced by pretreating the CD4- carrier with a proteolytic enzyme to expose a lipid layer.
21. A neutralization assay of claim 20, wherein said assay tests the neutralization activity of multispecific antibodies..
22. A method for treating HIV-1 infection, wherein said method comprises treating a patient in need of said treatment by administering to said patient an effective amount of a neutralizing antibody identified using the assay described herein.
3. A peptide produced by the method of assay of claim 1.
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EP2492279A1 (en) 2011-02-25 2012-08-29 Laboratorios Del. Dr. Esteve, S.A. Rapid immunogen selection method using lentiviral display
WO2012113921A1 (en) 2011-02-25 2012-08-30 Laboratorios Del Dr. Esteve, S.A. Rapid selection method for hiv gp-120 variants
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