WO2019186277A1 - Dosage de détection de p24 du vih-1 ultrasensible - Google Patents

Dosage de détection de p24 du vih-1 ultrasensible Download PDF

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WO2019186277A1
WO2019186277A1 PCT/IB2019/000386 IB2019000386W WO2019186277A1 WO 2019186277 A1 WO2019186277 A1 WO 2019186277A1 IB 2019000386 W IB2019000386 W IB 2019000386W WO 2019186277 A1 WO2019186277 A1 WO 2019186277A1
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hiv
beads
sample
fold
subject
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PCT/IB2019/000386
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English (en)
Inventor
Asier Saez-Cirion
Caroline PEREIRA BITTENCOURT PASSAES
Annie David
Darragh DUFFY
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Institut Pasteur
Institut National De La Sante Et De La Recherche Medicale
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Publication of WO2019186277A1 publication Critical patent/WO2019186277A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the invention is related to assays for detecting and quantifying HIV-1 p24 protein levels in biological samples and methods of using those assays in identifying and monitoring the presence of p24 in subjects and materials. Also provided are kits adapted for the use of the assays disclosed herein and methods of treating HIV in subjects that have been selected for treatment based on the application of the assays described herein.
  • HIV Human immunodeficiency virus
  • HIV-1 Optimal detection and monitoring of HIV-1 is critical for rapid diagnosis, early treatment initiation and ensure complete suppression of viremia and antigenemia while on antiretroviral treatment. Early treatment initiation is key to abort new HIV-1 transmission events. On the other hand, viral blips or incompletely suppressed viremia are associated with enhanced levels of harmful chronic inflammation in individuals receiving antiretroviral treatment.
  • the nucleocapsid protein HIV-1 p24 is a well-known surrogate for HIV-1 infection and viral replication. The lower the limit of detection of p24, the earlier a diagnosis of HIV-1 infection can be achieved and more efficiently the evolution of antigenimia can be monitored. Numerous variables can be manipulated to attempt to increase the sensitivity, standardization, and reproducibility of a biological molecule detection assay.
  • the assay includes the use of sandwich, competitive, direct, or indirect ELISA, the assay’s signal sensors (fluorescence, colorimetry, and others), the temperature and time of each reaction step, the pH, the presence or absence of ion chelators such as EDTA, the presence or absence of a surfactant or salts, the use of chaotropic agents, the treatment with proteases such as pepsin, the specificity and affinity of the target-binding antibodies that can be used in immunoassays, the concentration of the sample, the assay platform, and many others.
  • the effect of changing each of these parameters in the success of the assay is unpredictable and is determined empirically.
  • the present invention is directed to improved methods of detection and quantification of HIV-1 p24 and applications of such methods to monitoring HIV infections, identifying HIV-infected materials or subjects, methods of treatment of HIV in subjects selected for treatment based on the methods disclosed herein, kits for practicing the disclosed methods and other embodiments.
  • a method of quantitation of HIV p24 in a body fluid sample comprising: mixing the body fluid sample with an acidic solution to dissociate immune complexes in the resulting mixture; neutralizing the resulting mixture; contacting the neutralized resulting mixture with beads comprising one or more molecules that specifically bind HIV p24; and quantitating the p24 protein that is bound to the beads.
  • the method of quantitation is carried out in vitro.
  • the beads have an average diameter from about 0.1 micrometers to about 100 micrometers, from about 0.1 to about 10 micrometers, from about 0.1 to about 1 micrometer, from about 1 to about 10, or from about 1 micrometer to about 3 micrometers.
  • the beads are made from materials selected from plastics or synthetic polymers (e.g., polyethylene, polypropylene, polystyrene, polyamide, polyurethane, phenolic polymers, or nitrocellulose etc.), naturally derived polymers (latex rubber, polysaccharides, polypeptides, etc), composite materials, ceramics, silica or silica-based materials, carbon, metals or metal compounds (e.g., comprising gold, silver, steel, aluminum, copper, etc.), inorganic glasses, and silica.
  • plastics or synthetic polymers e.g., polyethylene, polypropylene, polystyrene, polyamide, polyurethane, phenolic polymers, or nitrocellulose etc.
  • naturally derived polymers latex rubber, polysaccharides, polypeptides, etc
  • composite materials ceramics, silica or silica-based materials, carbon, metals or metal compounds (e.g., comprising gold, silver, steel, aluminum, copper, etc.), inorgan
  • the antibody is selected from monoclonal antibodies, bispecific antibodies, minibodies, single domain antibodies, synthetic antibodies or antibody mimetics, chimeric antibodies, humanized antibodies, and antibody-drug conjugates.
  • the quantitation comprises using fluorescent-labeled antibodies, or fluorescent enzyme substrates, to detect p24 bound to the beads in the chambers.
  • the quantitation comprises using an enzyme conjugate to detect the biotinylated antibodies bound to the p24 in the chamber.
  • a method for monitoring HIV viral load in a subject comprising quantitating HIV p24 in the subject’s body fluid sample (preferably, plasma) over a period of time with the method according to any of the above embodiments. The method is performed in vitro.
  • a method of identifying the presence of HIV infection in a subject comprising quantitating HIV p24 in the subject’s body fluid sample (preferably, plasma) with the method according to any of the above embodiments.
  • the method is performed in vitro.
  • a method of treating a subject for HIV infection comprising administering an HIV inhibitor to a subject in need thereof, wherein the subject has been selected for treatment with a method according to any one of the previous embodiments.
  • a kit comprising an acidic solution for ICD, a neutralizing solution that neutralizes the acidic solution, beads, and a composition comprising a p24-binding molecule for coating or binding to the beads.
  • kit of embodiment 44 wherein the beads already comprise a p24-binding molecule.
  • kit of embodiment 45 wherein the p24-binding molecule is an antibody.
  • kits according to any one of embodiments 44 through 47 wherein the pH of the acidic solution is from 1 to 3, from 3 to 4, from 4 to 5, from 5 to 6, about 1 .5, or about 2.8.
  • the neutralizing solution comprises Tris.
  • the body fluid sample comprises a blood sample, serum sample, a plasma sample, or a depleted plasma sample, a semen sample, a sputum sample, an exudate, a saliva sample, a cerebrospinal fluid, or a tissue biopsy.
  • the body fluid sample comprises cells isolated from a subject (e.g., immune cells, cells isolated from cheeks or gums), aqueous humour, vitreous humour, bile, breast milk, endolymph, perilymph gastric juice, mucus, peritoneal fluid, pleural fluid, sebum, semen, sweat, tears, vaginal secretion, vomit, or urine.
  • FIG. 1 Frequency of H IV Gag p24 positive samples distributed by viral load range.
  • FIG. 2 Comparison of methods for immune complex dissociation and their impact in the Simoa® p24 assay. Bars denote the average enzyme per beads for: blank; 0 (2x); 0.01 ; 0.02; 0.07; 0.24; 0.67; 2.39 and 13.17 pg/mL of p24 STD non-treated (NT) and following various treatments.
  • FIG. 3 Impact of SDS and DTPA in Simoa® p24 quantification. Left. Comparison of standard curves treated (black symbols) or not (red symbols) with SDS/DTPA. Right. p24 quantification in different samples (including SDS/DTPA alone, the negative sample provided with the kit (STD A), and 10 plasmas from non-HIV infected donors.
  • FIG. 4 Validation of Glycine pH 1 8/Tris as an efficient method for immune complex dissociation. P24 levels determined by Simoa in plasmas from HIV-1 infected patients without treatment and after treatment with Glycine pH 1 8/Tris.
  • FIG. 5 Sensitivity of HIV p24 assay.
  • FIG. 6A Schematic representation of the protocol to concentrate plasma samples by ultracentrifugation.
  • FIG. 6B Current sensitivity of the ultrasensitive quantification by of p24 in plasma after Glycine/Tris treatment.
  • FIG. 7 p24 quantification in plasma samples (treated with Glycine/Tris) from 108 HIV-1 infected individuals at the time of cART initiation and after 24 and 48 weeks of treatment.
  • the term "about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 %, 0.05%, or 0.01 % of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • the terms “determining”, “assessing”, “assaying”, “measuring” and “detecting” refer to both quantitative and qualitative determinations, and as such, the term “determining” is used interchangeably herein with “assaying,” “measuring,” and the like. Where a quantitative determination is intended, the phrases “determining an amount” of an analyte and the like can be used. Where a qualitative and/or quantitative determination is intended, the phrase “determining a level" of an analyte or “detecting” an analyte is used.
  • control reference method is a reference method in which all of the parameters are identical to those of the method being compared with exception of the variable being tested.
  • binds an agent (e.g., antibody) that recognizes and binds a molecule (e.g., HIV p24), but which does not substantially recognize and bind other molecules in a sample, for example, a plasma sample.
  • an agent e.g., antibody
  • a molecule e.g., HIV p24
  • two molecules that specifically bind form a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity.
  • the term “specifically binds to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules inducting biological molecules.
  • an antibody that specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than if binds to other targets.
  • the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA).
  • an antibody that specifically binds to a target has a dissociation constant of (K D ) of ⁇ 1 x1 O 6 M, ⁇ 1 x1 O 7 M, ⁇ 1 x1 O 8 M, ⁇ 1 x10 9 M, or ⁇ 1 x1 O 10 M.
  • specific binding can include, but does not require exclusive binding.
  • subject is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline, preferably a human.
  • a human or non-human mammal such as a bovine, equine, canine, ovine, or feline, preferably a human.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 (or similarly a range between 1 and 50) is understood to include any number, combination of numbers, or sub-range from the group consisting 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • a range includes in particular the values framing said range.
  • Binding affinity generally refers to the strength of the sum total of non- covalent interactions between a single binding site of a molecule (e.g., of an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity”, “bind to”, “binds to” or “binding to” refers to intrinsic binding affinity that reflects a 1 :1 interaction between members of a binding pair (e.g., antibody Fab fragment and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ). For example, the lower the K D of an antibody, the lower the concentration of the antigen that needs to be present in the sample for the antibody to detect the antigen:
  • Most antibodies have K D values in the low micromolar (1 O 6 ) to nanomolar (10 7 to 10 9 ) range.
  • H igh affinity antibodies generally considered to be in the low nanomolar range (10 9 ) with very high affinity antibodies being in the picomolar (10 12 ) range.
  • Affinity can be measured by common methods known in the art, including those described herein.
  • Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.
  • the Label-free surface plasmon resonance (SPR)-based biosensors such as BIACORE methods, and MM/PBSA methods, and KinExA are standard methods often preferred. It is known that the binding affinities can change depending on the assay. Accordingly, for purposes of this disclosure, it is sufficient that the binding affinity fall within the recited range when measured by at least one method standard in the art.
  • the levels of p24 in biological samples are surrogates for the presence of HIV in a subject or material.
  • Aspects of the invention include methods that can be used to detect and/or measure or quantitate HIV p24 in biological samples from a subject. Immune complexes of p24 with anti-p24 antibodies can be produced during an immune response to HIV infection. The presence of these complexes interferes with the detection and quantification of p24 in biological samples. Aspects of the invention include methods that improve the detection and quantification of p24 in biological samples by dissociating some or all of the p24 in the sample from those immunological complexes, rendering it more accessible to detection.
  • Some of the methods of the invention comprise mixing a biological sample with an acidic solution to dissociate p24-containing immune complexes that might be present in the biological sample, neutralizing the resulting mixture after a period of immune complex dissociation (ICD), contacting the neutralized resulting mixture with beads comprising one or more molecules that specifically bind p24, and detecting the presence of and/or quantitating the p24 protein that is bound to the beads as a measure of the p24 protein that is present in the sample.
  • ICD immune complex dissociation
  • the mixing of an acidic solution with a biological sample is intended to result in a mixture having a pH between 1 .0 and 5.9, between 2.0 and 5.0, between 2.2 and 4.0, between 2.5 and 3.0.
  • the step of “neutralizing the resulting mixture” comprises the addition of a solution of basic pH (i.e., the“neutralizing solution”) to the resulting mixture so as to increase its pH to a pH 6.0 or 6.5, to a neutral pH, to a pH of 6.5 to 7.0, to a pH of 7.0 to 7.5, to a pH of 7.5 to 8.0, to a pH of 8.0 to 8.5, to a pH of 8.5 to 9.0, to a pH of 9.0 to 1 1 .0, or from a pH of 1 1 .0 to 14.0 .
  • the“neutralizing solution” a solution of basic pH
  • the biological sample comprises a body fluid sample such as a blood sample, serum sample, a plasma sample, or a depleted plasma sample, a semen sample, a sputum sample, an exudate.
  • the sample is obtained by blood draw.
  • the sample is obtained by finger-stick/prick or heel-prick.
  • the biological sample comprises an oral fluid sample.
  • the biological sample is a saliva sample.
  • the biological sample comprises cerebrospinal fluid or a tissue biopsy.
  • the biological sample comprises cells isolated from the subject (e.g., immune cells, cells isolated from cheeks or gums).
  • the biological sample is not directly from a subject but is derived from or comprises cells grown and/or processed in vitro.
  • the biological sample comprises aqueous humour, vitreous humour, bile, breast milk, endolymph, perilymph gastric juice, mucus, peritoneal fluid, pleural fluid, sebum, semen, sweat, tears, vaginal secretion, vomit, or urine.
  • the biological sample is a plasma sample.
  • the subject is a normal subject. In some embodiments, the subject has, or is suspected to have, an HIV infection. In some embodiments, the infection is acute. In some embodiments, the subject is in the acute stage because the subject is in the period from HIV acquisition until seroconversion. In some embodiments, the infection is chronic.
  • the subject is an adult. In some embodiments, the subject is a child. In some embodiments, the child is a newborn. In some embodiments, the child is less than 18 months old. [0033] In some embodiments, the subject has been, or is being exposed to anti- retroviral therapy. In some embodiments, the subject has been, or is being treated with one or more of the following HIV Inhibitors:
  • Entry inhibitors interfere with binding, fusion and entry of HIV-1 to the host cell by blocking one of several targets (Wikipedia).
  • Maraviroc works by targeting CCR5, a co-receptor located on human helper T-cells.
  • Enfuvirtide is a peptide drug that must be injected and acts by interacting with the N-terminal heptad repeat of gp41 of HIV to form an inactive hetero six-helix bundle, therefore preventing infection of host cells.
  • Nucleoside reverse transcriptase inhibitors and nucleotide reverse transcriptase inhibitors (NtRTI) are nucleoside and nucleotide analogues which inhibit reverse transcription.
  • NRTIs include zidovudine, abacavir, lamivudine, emtricitabine, and tenofovir.
  • NNRTI Non-Nucleoside reverse transcriptase inhibitors
  • 1 st generation NNRTIs include nevirapine and efavirenz.
  • 2nd generation NNRTIs include etravirine and rilpivirine.
  • Integrase inhibitors also known as integrase nuclear strand transfer inhibitors or INSTIs
  • Integrase inhibitors include raltegravir, elvitegravir, and dolutegravir.
  • Protease inhibitors block the viral protease enzyme necessary to produce mature virions upon budding from the host membrane.
  • HIV protease inhibitors are lopinavir, indinavir, nelfinavir, amprenavir, ritonavir, darunavir, and atazanavir.
  • Maturation inhibitors have a similar effect by binding to gag, and include bevirimat and becon.
  • Combination antiretroviral therapy is a mixture of at least two, and preferably three or more different classes of antiretroviral therapy. All different combinations of the antiretroviral therapies specified herein are specifically contemplated. Examples of cART include: [0041 ] Combivir: lamivudine + zidovudine.
  • Kaletra lopinavir + ritonavir
  • Trizivir abacavir + lamivudine + zidovudine
  • Epzicom in USA
  • Kivexa in Europe and Russia
  • lamivudine abacavir + lamivudine
  • Truvada tenofovir disoproxil fumarate + emtricitabine.
  • Atripla emtricitabine + tenofovir disoproxil fumarate + efavirenz
  • Complera in USA
  • Eviplera in Europe and Russia
  • Stribild elvitegravir + cobicistat + emtricitabine + tenofovir disoproxil fumarate.
  • Triumeq abacavir + dolutegravir + lamivudine.
  • Prezcobix darunavir + cobicistat.
  • Dutrebis lamivudine + raltegravir.
  • Genvoya elvitegravir + cobicistat + emtricitabine + tenofovir alafenamide fumarate.
  • Rev inhibitors interfere with the biogenesis of viral RNA required for the replication of HIV.
  • Rev inhibitor can function through binding to the Cap Binding Complex at the 5’ end of the mRNA coding for 3 structural proteins of the virus. By promoting HIV RNA splicing, these inhibitors can reduce the level of genomic RNA and inhibit HIV replication.
  • Preferred compounds can be found in U.S. Patents 9,145,367 and 9,061 ,999, which are hereby incorporated by reference. Particularly preferred compounds are 10-chloro-2,6-dimethyl-2H-pyrido [3’,4’:4,5]pyrrolo[2,3-g]isoquinoline (IDC16), 8-chloro-N-(4-(trifluoromethoxy)phenyl)quinolin-2-amine (ABX464) and 8- chloro-N-glucuronide-N-(4-(trifluoromethoxy)phenyl)quinolin-2 -amine) (ABX464-N- glucuronide) compounds, as set forth in Campos et al.
  • the immune complex dissociation is achieved by exposure of the biological sample (or a component thereof, which may have been diluted) to an acidic pH prior to quantitation or detection.
  • the acidic pH is reached by mixing the biological sample (or a component/dilution thereof) with an acidic solution.
  • the acidic solution comprises citric acidic or acetic acid (e.g., 300 mM).
  • the acidic solution comprises glycine.
  • the concentration of the glycine in the acidic solution is about 1 .5 M and the pH of the solution is anywhere from 1 to 6 (with HCI), for example, 1 .0-2.0, 2.0-3.0, 3.0-4.0, 4.0-5.0, 5.0-6.0, 6.0-7.0.
  • the pH of the glycine solution is about 1 .8.
  • the pH of the glycine solution is about 2.5.
  • the term about means plus or minus 0.05.
  • the acidic solution and the biological sample (or a component/dilution thereof) are mixed at a 1 :1 ratio (v/v).
  • the acidic solution is 0.15 M glycine-HCI with/without Triton X and it is added at a 1 :1 ratio to the biological sample (e.g., plasma). In some embodiments, the acidic solution is 0.15 M glycine-HCI and it is added at a 1 :2 ratio to the biological sample (e.g., plasma). In some embodiments, the acidic solution is GlycineHCI 1 .5 M _pH 1 .85 and the samples are diluted 1/3 in acidic solution. In other embodiments, the ratio of biological sample to acidic solution is a different ratio (v/v).
  • the ICD is carried out by incubation of the sample/acidic mixture at room temperature, or at 37°C. In some embodiments, the incubation is for 30 minutes at 37°C. In some embodiments, the incubation is for 1 hour at 37°C. In some embodiments, the incubation is for any time between 15 minutes and 90 minutes. In some embodiments, the ICD is achieved by combination of any of these acidic conditions with heat. In some of such embodiments, exposure to heat (in combination with acidic exposure) comprises incubating the sample/acidic mixture at 70°C for 10 minutes, or 4 minutes at 95°C.
  • the degree of ICD is measured. In some embodiments, the degree of ICD is not measured but some ICD is assumed to have taken place because of the acidic conditions. In some embodiments, the ICD is complete. In some embodiments, the ICD is partial. In some embodiments, the degree of ICD can be quantified by comparing the levels of p24 measured with and without ICD.
  • the neutralizing solution has a pH from 9 to 1 1 .
  • the neutralizing solution comprises TRIS base.
  • the pH is 9.0.
  • the pH of the TRIS base solution is 10.8.
  • the Tris is 1 M Tris.
  • the neutralizing solution is 1 M Tris, pH 9.5.
  • the neutralizing solution is 1 .5 M Tris HCI, pH 9.0.
  • the neutralizing solution and the sample/acidic mixture that has undergone partial or complete ICD are mixed at a ratio of 1 :2.
  • the neutralizing solution and the sample/acidic solution mixture are mixed at a ratio of 1 :3, 1 :4, 1 :5, 1 :10, 1 :100 or 2:1 , 3:1 , 4:1 , 5:1 , 10:1 , 100:1 .
  • the neutralizing solution is used to counteract the acidic pH of the solution in which the immune complex dissociation takes place.
  • the neutralized mixture is directly mixed with beads. In some embodiments, the neutralized mixture undergoes additional processing prior to incubation with the beads. In some embodiments, additional reagents are added to the neutralized mixture. In some embodiments, the additional reagents comprise a surfactant. In some embodiments, the surfactant is Triton X100.
  • the beads are magnetic. In some embodiments, the beads are not magnetic. In some embodiments, the beads are paramagnetic. In some embodiments, the beads average diameter from about 0.1 micrometers to about 100 micrometers, from about 0.1 to about 10 micrometers, from about 0.1 to about 1 micrometer, from about 1 to 10. In a preferred embodiment, the beads average diameter is from about 1 micrometer to about 3 micrometers.
  • the beads are of sphere-like shapes. In some embodiments, the beads are disks. In some embodiments, the beads are rings. In some embodiments, the beads have cube-like shapes. In some embodiments, the beads have a combination of shapes.
  • the beads are made from materials selected from plastics or synthetic polymers (e.g., polyethylene, polypropylene, polystyrene, polyamide, polyurethane, phenolic polymers, or nitrocellulose etc.), naturally derived polymers (latex rubber, polysaccharides, polypeptides, etc), composite materials, ceramics, silica or silica-based materials, carbon, metals or metal compounds (e.g., comprising gold, silver, steel, aluminum, copper, etc.), inorganic glasses, silica, or a combination thereof.
  • plastics or synthetic polymers e.g., polyethylene, polypropylene, polystyrene, polyamide, polyurethane, phenolic polymers, or nitrocellulose etc.
  • naturally derived polymers latex rubber, polysaccharides, polypeptides, etc
  • composite materials ceramics, silica or silica-based materials, carbon, metals or metal compounds (e.g., comprising gold, silver, steel, aluminum, copper,
  • the beads are partially (e.g., 1 %, 5%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or any values or ranges in between) coated by or conjugated to another material. In some embodiments, the beads are completely or about completely coated by or conjugated to another material. In some embodiments, the beads are coated by or conjugated to p24-binding molecule(s). In some embodiments, the coating or conjugation are done directly. In some embodiments, the coating or conjugating are indirect (e.g., there is another intermediate molecule between the beads and the p24-binding molecule). In some embodiments, the beads are coated or conjugated to p24-binding molecules of a single type. In some embodiments, there is more than one type of p24-binding molecule on the beads.
  • the beads have approximately 250,000 p24- binding sites per bead or fewer. In some embodiments, the beads have between 50,000 and 300,000 binding sites per bead. In some embodiments, the beads have between 5,000 and 50,000 p24-binding sites per bead. In some embodiments, the beads comprising the p24-binding can be prepared by means described in, for example, U.S. patent application Ser. No. 12/731 ,130, entitled "Ultra-Sensitive Detection of Molecules or Particles using Beads or Other Capture Objects" by Duffy et al., filed Mar. 24, 2010; and International. Patent Application No.
  • PCT/US1 1/026645 entitled “Ultra-Sensitive Detection of Molecules or Particles using Beads or Other Capture Objects” by Duffy et al., filed Mar. 1 , 201 1 , each herein incorporated by reference).
  • the p24-binding molecule is a protein. In some embodiments, the p24-binding molecule is a polypeptide. In some embodiments, the p24-binding molecule is a peptide.
  • the p24-binding molecule is an antibody.
  • the antibody is a monoclonal antibody, polyclonal antibody, bispecific antibody, minibody, single domain antibody, nanobody, synthetic antibody or antibody mimetic, chimeric antibody, humanized antibody, or antibody-drug conjugate.
  • the antibody is a human antibody, a murine antibody, a rabbit antibody, goat antibody, or a horse antibody.
  • the p24-binding molecule is an antibody fragment.
  • the p24-binding antibody fragment is an Fab, Fab', (Fab')2, Fv, or scFv fragment.
  • the p24-binding molecule binds p24 with an affinity between 10 5 -10 10 M- 1 .
  • the binding constant between the p24- binding molecule and p24 is from at least about 10 4 to about 10 6 M- 1 , at least about 10 5 to about 10 9 M- 1 , at least about 10 7 to about 10 9 M- 1 , greater than about 10 9 M- 1 .
  • neutralized solution i.e., comprising the biological sample
  • the beads with or without some additional reagent(s)
  • the mixture of the neutralized solution and the beads is processed through a Simoa® p24 assay obtained from Quanterix®.
  • the p24 in the mixture is detected or quantitated by a Quanterix® Simoa® detector such as Simoa® FID-1 or a Simoa® SR-X.
  • the Simoa® p24 assay obtained from Quanterix® does not use immune complex dissociation.
  • the assay is used on samples (e.g., plasma samples) that are not previously subjected to any ICD process, including no acid-base dissociation.
  • the assay is sold as a kit and the kit does not comprise any reagents for immune complex dissociation or any instructions recommending or suggesting that ICD be applied to the biological samples prior to processing via the assay.
  • the typical Simoa® p24 assay obtained from Quanterix® comprises the following reagents: Bead reagent, detector reagent, SBG reagent, Sample diluent, calibrators, controls, and RGP reagent.
  • the lower limit of detection is advertised as 0.0027 pg/mL and the lower limit of quantification of 0.010 pg/mL, using a Simoa® FID- 1 Analyzer.
  • the typical sample volume is 154 microliters.
  • Simoa Immunoassays have either 2 or 3 incubation steps: [0075] In a 2-step immunoassay, target antibody coated paramagnetic beads are combined with sample and biotinylated detector antibody in the same incubation. Target molecules present in the sample are captured by the antibody coated beads and bind with the biotinylated antibody detector simultaneously.
  • target antibody coated paramagnetic beads are combined and incubated with sample alone.
  • Target molecules present in the sample are captured by the antibody coated beads.
  • biotinylated detector antibodies are mixed and incubated with the beads. The detector antibodies bind to the captured target during this additional incubation.
  • SBG streptavidin-3 ⁇ 4-galactosidase
  • the beads are resuspended in a resofurin b-D galactopyranoside (RGP) substrate solution and transferred to the Simoa Disc. Individual beads are then sealed within microwells in the array. If the target has been captured and labeled on the bead, b-galactosidase hydrolyzes the RGP substrate in the microwell into a fluorescent product that provides the signal for measurement.
  • RGP resofurin b-D galactopyranoside
  • a single-labeled target molecule results in sufficient fluorescent signal in 30 seconds to be detected and counted by the Simoa optical system.
  • the percentage of bead-containing wells in the array that have a positive signal is proportional to the amount of target present in the sample.
  • the total fluorescence signal is proportional to the amount of target present in the sample.
  • the detection and/or quantitation step is done by “single molecule array” technology.
  • the“single molecule array technology” is a technology originally described in Rissin DM et al. 2010. Single- molecule enzyme linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol 28:595-599. http://dx.doi.org/10.1038/nbt.1641 ; Rissin et al. 201 1 . Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range. Anal Chem 83:2279-2285.
  • the “single molecule array” technology is that essentially described in Wilson, D.H. et al.
  • the Simoa® HD-1 Analyzer A Novel Fully Automated Digital Immunoassay Analyzer with Single-Molecule Sensitivity and Multiplexing, Journal of Laboratory Automation 2016, Vol. 21 (4) 533-547 or routine modifications of the same.
  • the “single molecule array” technology is that essentially described in Cabrera et al. Rapid, Fully Automated Digital Immunoassay for p24 Protein with the Sensitivity of Nucleic Acidic Amplification for Detecting Acute HIV Infection, Clinical Chemistry 2015, 61 :1 1 , 1372-1380; or in Kelley, S.O., et al. Advancing the speed, sensitivity and accuracy of biomolecular detection using multi-length-scale engineering, Nat Nanotechnol. 2014 December ; 9(12): 969- 980. doi:10.1038/nnano.2014.261 .
  • the“single molecule array” technology is the Simoa® HIV P24 KIT sold by Quanterix®.
  • the detection and/or quantitation step is done by a digital assay. In some embodiments, the detection and/or quantitation step is done by a combination of a digital assay with an analog assay. In some embodiment, the detection and/or quantitation step is done by an analog assay.
  • the sensitivity of the claimed quantitation method is increased at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 40 fold, at least 50 fold, at least, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 500 fold, at least 1000 fold, over the sensitivity of a control reference quantitation method identical to the claimed method but without the claimed acidic-based ICD.
  • the improve in sensitivity is measured against that of another reference quantitation method.
  • the sensitivity of the claimed quantitation method is increased at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 40 fold, at least 50 fold, at least, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 500 fold, at least 1000 fold over the sensitivity of a reference quantitation method without heat-based ICD (with and without SDS/DTPA), SDS/DTPA, and/or acidic-based ICD.
  • the claimed method can detect a viral load of 10 3 , 10 4 , 10 5 , or 10 6 viruses/ml in a plasma sample.
  • claimed method has a sensitivity of at least 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or 200 fg/ml of p24. In some embodiments, the claimed method can detect, at least, or as low as, between 20-30, 20-40, 20-80, 20-150 fg/ml of p24.
  • the claimed method has a sensitivity of at least or as low as 24 fg of p24 per ml_ of plasma.
  • the claimed assay is capable of detecting acute HIV infection as early or earlier than commercial Nucleic Acidic Testing (e.g., Roche HIV RNA CAP/CTM assay, Siemens HIV RNA bDNA assay, Abbott HIV RNA).
  • commercial Nucleic Acidic Testing e.g., Roche HIV RNA CAP/CTM assay, Siemens HIV RNA bDNA assay, Abbott HIV RNA.
  • the assay is capable of detecting HIV infection as early as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 14, 21 , 28 days after infection.
  • the claimed method detects HIV p24 in at least 25%, at least 50%, at least 80%, at least 85%, at least 90%, or at least 95% of the plasma samples with a viral load below 10 6 copies/mL, or below 10 5 , below 10 4 , equal to or below 10 3 .
  • the claimed method detects HIV p24 in at least 25%, at least 50%, at least 80%, at least 85%, at least 90%, or at least 95% of the plasma samples with a viral load equal to or below 10 3 copies/mL.
  • the detection and quantitation methods of the invention can be used to monitor HIV viral load in subjects. In some embodiments, the detection and quantitation methods are any of the methods described in the previous sections. [0094] In one embodiment, the detection and quantitation methods of the invention can be used to identify or detect the presence of HIV infection in a subject. In some embodiments, the detection and quantitation methods are any of the methods described in the previous sections.
  • the detection and quantitation methods of the invention can be used to select a subject for HIV treatment. Accordingly, one aspect of the invention is a method of treating HIV infection by selecting a patient for treatment with any one of the methods described herein and administering an HIV inhibitor to the patient.
  • the monitoring is done to assess treatment efficacy or monitor the response to a medication in a subject in need thereof. In one embodiment, the monitoring is done to assess the eradication of HIV viral infection. In one embodiment, the monitoring/quantification/detection of the HIV p24 in the subject is done to help design a drug dosage regimen. In one embodiment, the monitoring/quantification/detection of the HIV p24 is done to predict immunologic response of a subject to antiretroviral therapy, to identify the stage of the infection or an HIV-related disease, or to monitor the progression of the infection or of an HIV-related disease (e.g., HIV-1 -associated neurocognitive disorder)
  • HIV-related disease e.g., HIV-1 -associated neurocognitive disorder
  • the invention is also directed to a kit adapted for carrying out the methods of the invention wherein the kit comprises one or more of the components of the instruments, containers, reagents, standards, buffers and the like that are utilized in and optimized for the practice of the methods of the invention.
  • the components of the kit may be packaged or compartmented separately from the rest of the components.
  • the kit comprises an acidic solution for ICD, a neutralizing solution that neutralizes the acidic solution, and beads comprising a p24- binding molecule.
  • the acidic solution, the neutralizing solution, and the beads are as described in the previous sections.
  • the kit further comprises an internal control or standard for p24 quantification and/or for preparation of a calibration curve.
  • the beads and the p24-binding molecule are provided in separate containers of the kit.
  • the kit further comprises reagents for detection of the p24 that binds to the beads.
  • reagents are antibodies (themselves labeled or unlabeled).
  • the kit further comprises substrates for enzymes that are used to detect the p24 that is bound to the beads.
  • the substrate emits a fluorescent signal.
  • the substrate emits a colorimetric signal.
  • the p24 levels in plasma samples of HIV infected individuals with different levels of viremia were determined. A total of 31 plasma samples obtained from different cohorts followed at Necker Hospital (Table 1 ) were analyzed. Samples of individuals in acute and chronic HIV infection were selected to be tested. For this first assay, plasma samples were thawed at room temperature, centrifuged at 3000rpm for 5 minutes, then plated in a Simoa® plate (200mI) and inactivated with Triton (20mI Triton 20%). p24 quantification was performed as recommended by the manufacturer of the HIV p24 detection kit HIV p24 102215 (Quanterix®).
  • Table 1 Plasma samples from HIV infected patients with different viral load levels to test the sensitivity of HIV Gag p24 assay.
  • results obtained are depicted in FIG. 1.
  • HIV p24 could be detected in 100% of samples with viral load ranging from 10 6 -10 7 copies/mL. However, the p24 in samples with lower viral loads was not efficiently detected (see decreased frequency of p24 positive samples in FIG. 1 ). Due to the high sensitivity previously observed and described for this assay, it would have been expected to observe a higher frequency of p24 positive samples.
  • Assays were done using the standard samples provided with the HIV p24 kit. Standards (STD) were thawed, and 100mI were transferred to a new tube. The different treatments were done as follows:
  • the p24 plasma levels were significantly higher before treatment initiation and strongly decreased upon treatment initiation, as expected. These results confirmed that ultrasensitive quantification of p24 in plasma levels following the acidic- based ICD protocol described herein allow monitoring changes in p24 levels in individuals in chronic HIV-1 infection. Interestingly, p24 was still detectable in some samples after 48 weeks of treatment, which could have important implications for the care of these individuals.
  • HIV 1 -positive patients before and after immune-complex dissociation a study of a 5- year period, Clin Diagn Virol. 1995 Feb;3(2):131 -7.

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Abstract

L'invention concerne des dosages pour détecter et quantifier les taux de protéine p24 du VIH-1 dans des échantillons biologiques et des procédés d'utilisation de ces dosages dans la surveillance et l'identification de la présence de p24 dans des sujets et des matériaux. L'invention concerne également des kits conçus pour l'utilisation des dosages selon l'invention et des procédés de traitement du VIH chez des sujets qui ont été sélectionnés pour un traitement sur la base de l'application des dosages selon l'invention.
PCT/IB2019/000386 2018-03-28 2019-03-28 Dosage de détection de p24 du vih-1 ultrasensible WO2019186277A1 (fr)

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WO2023088444A1 (fr) * 2021-11-20 2023-05-25 东莞市朋志生物科技有限公司 Anticorps contre le vih-1 p24, son procédé de préparation et son utilisation

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