Method for evaluating sensitivity of virus to treatment by measuring enzyme activity and system thereof
阅读说明：本技术 用于通过测量酶活性来评估病毒对治疗的敏感性的方法及其系统 (Method for evaluating sensitivity of virus to treatment by measuring enzyme activity and system thereof ) 是由 斯蒂娜·恩奎斯特 克拉斯·卡兰德 汤米·加图 安德斯·马尔姆斯滕 英瓦尔·彼得松 于 2018-07-03 设计创作，主要内容包括：本发明涉及用于评估病毒对用抑制野生型病毒中酶的药物进行的治疗的敏感性的方法，其包括以下步骤：a)从包含病毒的样品提取病毒酶；b)在不存在药物的情况下和在存在单个预先确定的浓度的药物的情况下测量病毒酶活性；以及c)由在存在药物的情况下和在不存在药物的情况下酶活性之间的关系来确定病毒对用药物进行的治疗的敏感性。本发明还涉及用于执行所述方法的系统。(The present invention relates to a method for assessing the sensitivity of a virus to treatment with an agent that inhibits an enzyme in a wild-type virus, comprising the steps of: a) extracting viral enzymes from a sample comprising a virus; b) measuring viral enzyme activity in the absence of the drug and in the presence of a single predetermined concentration of the drug; and c) determining the susceptibility of the virus to treatment with the drug from the relationship between the enzyme activity in the presence of the drug and in the absence of the drug. The invention also relates to a system for carrying out said method.)
1. A method for assessing the sensitivity of a virus to treatment with an agent that inhibits an enzyme in a wild-type virus, comprising the steps of:
a) extracting the viral enzyme from a sample comprising said virus;
b) measuring viral enzyme activity in the absence of the drug and in the presence of a single predetermined concentration of the drug; and
c) determining the susceptibility of the virus to treatment with the drug from the relationship between the enzyme activity in the presence of the drug and in the absence of the drug.
2. A method for assessing whether a patient being treated for a viral infection requires alteration of a drug treatment with a drug that inhibits a viral enzyme due to resistance of the virus to said drug treatment, comprising assessing the sensitivity of the virus to treatment with said drug according to claim 1, wherein a sensitivity below a predetermined cut-off level is indicative of the need to alter the drug treatment.
3. A method for determining the load of a virus in a patient sample and the resistance of said virus to treatment with a drug that inhibits an enzyme in a wild-type virus, said method comprising the steps of:
a) extracting the enzyme from a sample comprising the virus;
b) dividing the extracted enzyme into at least two aliquots: a first aliquot and a second aliquot;
c) measuring the enzyme activity in the first aliquot in the absence of the drug and measuring the enzyme activity in the second aliquot in the presence of a single predetermined concentration of the drug;
d) providing a series of standard enzyme activity values correlating enzyme activity to viral load;
e) determining the viral load in the sample from the enzyme activity in the absence of the drug based on the enzyme activity standard value; and
f) viral resistance to treatment with the drug is determined from the relationship between enzyme activity in the presence and absence of the drug, respectively.
4. The method of any one of claims 1 to 3, wherein the virus is a retrovirus and the enzyme is a reverse transcriptase packaged into the retrovirus.
5. The method according to claim 4, wherein the retrovirus is a lentivirus, such as HIV, SIV, FIV, β -retrovirus, such as JSRV or MMTV, delta-retrovirus, such as BLV, HTLV-1 or HTLV-2, or gamma-retrovirus, such as PERV or MMuLV.
6. The method of claim 4 or 5, wherein the drug is a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a Nucleoside Reverse Transcriptase Inhibitor (NRTI).
7. The method of claim 6, wherein the NNRTI drug is selected from the group consisting of: nevirapine, Efaviren, rilpivirine, etravirine, delavirdine, rivvirine, GSK 2248761, RDEA806, BILR 355BS, calanolide A, MK-4965, MK-1439, MK-6186, doravilin and efavirine.
8. The method of claim 6, wherein the NRTI drug is selected from the group consisting of: zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, tenofovir fumarate, susafovadine, MK-8591, adefovir, telbivudine, and entecavir.
9. The method of any one of the preceding claims, wherein the predetermined drug concentration corresponds to the IC for a reference enzyme with a known level of drug resistance50The value is obtained.
10. The method of any one of claims 1 to 8, wherein the predetermined concentration corresponds to the IC of the drug against the wild-type enzyme5010 to 50 times the value, e.g. IC of the drug against the wild-type enzyme5015, 20, 25, 30, 35, 40 or 45 times the value.
11. The method of any one of the preceding claims, wherein the sample is a blood sample, a serum sample, a plasma sample, a viral preparation from cell culture, milk, saliva, semen, genital secretions, urine, intraperitoneal fluid, or cerebrospinal fluid.
12. A system for performing at least steps c) to f) of the method according to any one of claims 3 to 11, comprising a computer and a device for determining enzyme activity in a sample, the device being configured to transmit enzyme activity values to the computer, and wherein the computer is configured to receive the enzyme activity values and a series of standard values relating enzyme activity values to viral load, and is programmed to determine the viral load in the sample from the enzyme activity in the absence of the drug based on the standard values relating enzyme activity values to viral load; and determining the resistance of the virus to treatment with the drug from the relationship between enzyme activity in the presence of the drug and in the absence of the drug.
13. The system of claim 12, wherein the means for determining enzyme activity in a sample further comprises means for automatically extracting viral enzyme from a sample.
Technical Field
The present invention relates to biological assays and in particular to methods for determining viral drug sensitivity (susceptability) of a virus strain infecting an individual and the viral load in an individual. The invention also relates to a system for performing such a method.
Background
The main objective of antiretroviral therapy (ART) is to permanently suppress the positive replication of plasma viruses to undetectable levels, thereby delaying disease progression and prolonging survival. Currently in a resource-limited context, treatment of HIV-1 relies on a combination of two nucleosides with a non-nucleoside reverse transcriptase inhibitor (NNRTI) (nevirapine (NVP) or efavirenz (efavirenz)) as first-line therapy (Neogi et al 2013). Successful treatment outcomes require extended accessibility and close monitoring of ART. In a high revenue context, this is achieved by quantitative viral load monitoring every 3 to 6 months, as viral load monitoring detects treatment failure (Bryant 2013). Early detection of virus failure provides an opportunity to enhance compliance coaching to increase compliance with ART, potentially leading to re-suppression of viral load before resistant virus evolution can occur.
The currently accepted marker for viral load is the measurement of HIV RNA in plasma. This can be done by PCR, NASBA or by branched DNA techniques (see Revets et al 1996). All of these assays are based on amplification of HIV-1 virion RNA, which is considered impractical for large scale use in resource-limited settings because it requires infrastructure, facilities for molecular diagnostics, expensive equipment and skilled technicians, which are often unavailable.
An alternative to measuring HIV-1RNA is to measure the activity of the viral Reverse Transcriptase (RT). A less technically demanding assay for measuring RT enzyme activity using an enzyme linked immunosorbent assay (ELISA) based method has been developed by Cavidi, AB, Sweden, which has shown promising results (Labbett 2009, Huang 2010, Gupta 2016). The method used consisted of the following steps: I) the host polymerase activity present in the sample is inactivated without affecting the viral enzymes present in the enveloped virus particles (virion) (PCT/SE 002/00612). II) removing the enzyme inactivator, the enzyme activity blocking antibody, the endogenous enzyme activity inhibitor and the antiviral drug. III) extracting the concentrated purified virus RT (PCT/SE 01/00617). IV) RT activity was quantified using a sensitive enzyme assay (PCT/EP 00/05563).
The choice of HIV-1 resistance in those with first-line ART failures limits second-line and future treatment options. Therefore, antiretroviral resistance testing is important in the long-term management of HIV-1 infection. Because traditional phenotypic resistance assays are time consuming, expensive and require specialized laboratory facilities, genotypic resistance tests are often used. However, there are many obstacles to using genotypic resistance tests in low-income countries, such as cost and the need for specific equipment. Therefore, other methods are sought. An alternative is to determine phenotypic viral susceptibility at the RT enzyme level. It is desirable to characterize enzymes that have been extracted directly from viruses circulating in a patient's blood. The benefit of such a method is that the sample will reflect the positive replicating viral population in the patient at the time the blood sample was taken.
Failure of first-line antiretroviral therapy (ART) in 10% to 30% of treated patients may be due to drug resistance, drug toxicity, reduced compliance, and discontinuation of therapy transmitted or acquired (Goodall et al 2014, Hamers2012a, b). Thus, analysis of phenotypic susceptibility to RT inhibitory drugs is potentially an affordable test to timely distinguish drug resistance from reduced compliance. This concept has been explored by resistance testing with PCR-based Amp RT assay (US581745, US 5849494).
Another assay based on a different technical development line is the drug sensitivity assay developed by Cavidi AB. This method carries out a resistance test on enzymes extracted directly from patient plasma samples. It is based on a similar method used in the Cavidi HIV VL assay described above. Virus was purified by ion exchange chromatography and the drug sensitivity profile of the extracted viral enzymes was determined by IC in a sensitive enzyme assay50Values were characterized (PCT/SE02/01156, Shao et al 2003).
Although potentially useful, these methods have to date had limited practical significance in the management of HIV pandemics. The Amp RT assay is very sensitive, but at best is semi-quantitative, and it is also difficult to design controls to take into account the discrepancies that occur during initial reverse transcription. The disadvantage of the Cavidi method is that it is labor intensive and has a long turnaround time. The need to analyze each extracted enzyme in the presence of several drug dilutions also increases the total amount of enzyme required and compromises test sensitivity.
Summary of The Invention
The present invention is a technically simple robust test that provides comprehensive information on both HIV Viral Load (VL) and susceptibility to antiretroviral therapy (ART). The assay is amenable to automation, has a reduced turnaround time and requires that each enzyme sample extracted be analyzed in the presence of only one predetermined drug concentration.
The invention is based on the idea that: to assess drug susceptibility in patients already infected with a virus (e.g., HIV) and treated with a drug that inhibits the enzyme produced by the infected virus, the activity of the enzyme can be determined in the absence of the drug and in the presence of a single predetermined drug concentration of the drug, and the enzyme activities in the presence and absence of the drug, respectively, compared to obtain an assessment of drug susceptibility. This is an advantage over the prior art as it significantly reduces the number of enzyme activity assays that must be performed at serial dilutions of the drug when using the prior art. Measurement of enzyme activity in the absence of drug can also be used to determine the viral load of a patient, further increasing the amount of information that can be obtained in a single assay run.
It is therefore an object of the present invention to provide an improved test method for integrating drug susceptibility of a virus recovered from an individual with the viral load of the virus.
Accordingly, the present invention provides a method for assessing the susceptibility of a virus to treatment with an agent that inhibits an enzyme in a wild-type virus, comprising the steps of:
a) extracting viral enzymes from a sample comprising a virus;
b) measuring viral enzyme activity in the absence of the drug and in the presence of a single predetermined concentration of the drug; and
c) the sensitivity of a virus to treatment with a drug is determined by the relationship between the enzyme activity in the presence of the drug and in the absence of the drug.
The present invention also provides a method for assessing whether a patient being treated for a viral infection needs to be treated with an altered enzyme inhibiting drug due to viral resistance to the drug used for the treatment, comprising the steps of:
a) extracting viral enzymes from a patient sample comprising a virus;
b) measuring viral enzyme activity in the absence of the drug and in the presence of a predetermined concentration of the drug; and
c) the sensitivity of the virus to treatment with the drug is determined by the relationship between the enzyme activity in the presence of the drug and in the absence of the drug,
wherein sensitivity below a predetermined cut-off level indicates a need to change drug therapy.
The invention also provides a method for determining the load of a virus in a patient sample and the resistance of said virus to treatment with a drug inhibiting an enzyme in a wild-type virus, said method comprising the steps of:
a) extracting an enzyme from a sample comprising a virus;
b) the extracted enzyme was divided into at least two aliquots (aliquot): a first aliquot and a second aliquot;
c) measuring the enzyme activity in the first aliquot in the absence of the drug and measuring the enzyme activity in the second aliquot in the presence of a single predetermined concentration of the drug;
d) providing a series of standard values correlating enzyme activity to viral load;
e) determining the viral load in the sample from the enzyme activity in the absence of the drug based on the enzyme activity standard value; and
f) viral resistance to treatment with a drug is determined by the relationship between enzyme activity in the presence and absence of the drug, respectively.
According to this method, for each patient sample recovered, the activity of the enzyme is measured in the absence of the drug and in the presence of a single drug concentration, respectively. The enzyme activity recovered in the absence of drug was then recalculated into viral load and the ratio between the enzyme activities in the presence and absence of drug, respectively, was determined to provide information on the sensitivity to the antiviral drug.
The invention also provides a system for performing at least steps c) to f) of the above method, the system comprising a computer and a device for determining enzyme activity in a sample, the device being configured to transmit enzyme activity values to the computer, and wherein the computer is configured to receive the enzyme activity values and a series of standard values relating enzyme activity values to viral load, and is programmed to determine the viral load in the sample from the enzyme activity in the absence of a drug based on the standard values relating enzyme activity values to viral load; and determining virus resistance to treatment with the drug from the relationship between enzyme activity in the presence and absence of the drug.
Thanks to the solution according to the invention, a phenotypic test is achieved that can be used to evaluate the effect of a drug on enveloped viruses (e.g. retroviruses) present in a patient sample (e.g. plasma). This test (also referred to as an "assay") provides a method for combined viral quantitation and phenotypic resistance testing of viral enzymes recovered directly from patient samples. Viral load and drug resistance were measured in two aliquots derived from the same patient sample. Preferably the measurements are made in parallel (e.g., simultaneously or substantially simultaneously). Thus, valuable information on both viral load and drug sensitivity can be obtained from a single run of one biological sample in a time efficient manner. This is beneficial to the patient as it may provide rapid and educated guidance to the medical practitioner in additional pharmacotherapeutic treatments. In other words, the method and system according to the present invention provide reliable results (achievable within only one working day) within a rather short time span, so that a medical practitioner can quickly judge the patient status and the appropriate continuous treatment path, e.g. change of medication due to the occurrence of viral resistance.
According to one aspect of the invention, the virus is an enveloped virus.
According to one aspect of the invention, the virus is a retrovirus and the enzyme is a Reverse Transcriptase (RT) packaged into said retrovirus. Accordingly, one aspect of the present invention relates to a method of combined testing for viral load and phenotypic drug susceptibility in a virally infected mammalian subject by testing for RT enzymes packaged into enveloped viruses recovered from a biological sample (e.g., blood or plasma) from the subject.
In various embodiments, wherein the retrovirus is a lentivirus, such as HIV, SIV, FIV, β -a retrovirus, such as JSRV or MMTV, a delta-retrovirus, such as BLV, HTLV-1 or HTLV-2, or a gamma-retrovirus, such as PERV or MMuLV.
According to one aspect of the invention, the drug is a non-nucleoside reverse transcriptase inhibitor (NNRTI). Many NNRTI drugs are known, such as nevirapine, efavirenz, rilpivirine, etravirine, delavirdine, lersivirine, GSK 2248761, RDEA806, BILR 355BS, calanolide A, MK-4965, MK-1439, and MK-6186(Usach et al, 2013), doraviline, and efavirenz (elsufavirine).
According to one aspect of the invention, the drug is a Nucleoside analog reverse Transcriptase Inhibitor (NRTI). Many NRTI drugs are known, such as zidovudine (zidovudine) (AZT/3 '-azido-2', 3 '-dideoxythymidine), didanosine (didanosine) (ddI/2' -3 '-dideoxyinosine), zalcitabine (zalcitabine) (ddC/2' -3 '-dideoxycytidine), stavudine (stavudine) (d 4T/2', 3 '-didehydro-2', 3 '-dideoxythymidine), lamivudine (lamivudine) (3/(-) -L-2', 3 '-dideoxy-3' -thiacytidine), abacavir (abacavir) (ABC/[ (1S, 4R) -4- [ 2-amino-6- (cyclopropylamino) purin-9-yl ] cyclopent-2-en-1-yl ] methanol), Emtricitabine (emtricitabine) (FTC/2 ' -deoxy-5-fluoro-3 ' thiacytidine), Tenofovir Disoproxil Fumarate (TDF), sucravidine (censvudine, 4 ' -ethynyl-d 4T), MK-8591(EFdA/4 ' -ethynyl-2-fluoro-2 ' -deoxyadenosine), adefovir (adefovir), telbivudine (telbivudine), and entecavir (entecavir).
According to another aspect of the invention, the predetermined drug concentration corresponds to the IC for a reference enzyme with a known drug resistance level50The value is obtained.
The predetermined drug concentration may also correspond to the IC of the drug against the wild-
In one aspect of the invention, the predetermined drug concentration corresponds to a clinically relevant cut-off value, e.g. the concentration that gives the greatest distinction between sensitive and resistant viruses.
According to another aspect of the invention, the patient sample is a blood sample, a serum sample, a plasma sample, a virus preparation from cell culture, milk (break milk), saliva, semen, genital secretions (genetic secretion), urine, intra-peritoneal fluid (intraperitoneal fluid) or cerebrospinal fluid.
Also provided within the scope of the invention is a method for assessing whether a patient being treated for a viral infection needs to change drug therapy due to viral resistance to a drug used for treatment, comprising the method according to
Extraction of viral enzymes can be achieved in a variety of ways. In one embodiment, the extracting comprises: the viral particles of the sample are isolated and then lysed to release the viral enzyme (reverse transcriptase in the case of HIV) for further assay. The viral load and drug sensitivity profiles of individuals are determined in parallel by the recovered enzyme using sensitive enzyme assays, as will be described in more detail later.
The described enzyme isolation technique can be used for any retrovirus, but in the present application the method is exemplified by virus quantification and drug resistance testing using plasma-derived lentivirus RT.
In the following, the present invention is exemplified by several different retroviruses and the corresponding retrovirus-encoded enzyme Reverse Transcriptase (RT), but it will be understood that the methods and systems of the present invention are also applicable to other types of retroviruses comprising RT enzyme. The invention is also exemplified by a specific method for measuring enzyme activity.
Brief Description of Drawings
FIGS. 1A to 1B show a schematic step-by-step overview of an assay according to one example of the invention;
FIG. 2 shows an overview of virus isolation steps according to one example of the invention;
FIG. 3 shows an overview of a wash step according to one example of the invention;
FIG. 4 shows an overview of the RT extraction steps according to one example of the invention;
FIG. 5 shows an overview of the RT reaction steps according to one example of the invention;
FIG. 6 shows an overview of the conjugate binding step according to one example of the invention;
FIG. 7 shows an overview of the substrate reaction steps according to one example of the invention;
FIG. 8A shows inhibition curves and IC for wt and mutant controls in a drug resistance assay according to an example of the invention50An example of a value;
FIGS. 8B to 8C show the inhibition curves of FIG. 8A, highlighting inhibition at an EFV concentration of 10 μ M;
FIG. 9 illustrates a drug addition step in a drug resistance indication assay according to an embodiment of the present invention;
FIG. 10 shows an example of residual activity results of a drug resistance indicator assay according to an example of the present invention;
FIG. 11 shows an example of a layout of RT reaction plates according to the invention;
FIG. 12 shows an IC50One example of a relationship with remaining RT activity;
FIG. 13 shows an example of the correlation between the PhenoSense fold increase and the residual RT activity from assays according to the invention; and
FIG. 14 shows the relationship between HIV VL determined using real-time PCR with Abbott m2000rt and RTa determined using the assay of the present invention.
Fig. 15 is a schematic diagram of a system according to the present invention.
FIG. 16 is a diagram of one embodiment of a portion of a system according to the present invention.
Detailed Description
One way of implementing the invention will now be described mainly with reference to fig. 1A and 1B, which show an overview of the method steps to be performed. Here, the extraction of viral enzymes is schematically shown, and subsequent parallel, simultaneous determination of the viral load and drug susceptibility profile of an individual by recovered enzymes by using sensitive enzyme assays.
Fig. 1A shows steps I) to IV) and fig. 1B shows steps V) to VII) summarized as follows:
I) HIV purification: providing a biological sample, adding an enzyme inactivating agent for inactivating polymerase activity other than polymerase activity present in enveloped virus particles, and subsequently removing the enzyme inactivating agent, enzyme activity blocking antibody, endogenous enzyme activity inhibitor, and antiviral drug;
II) reverse transcriptase extraction: lysing the viral particles to release the enzyme and recovering the concentrated purified viral enzyme from the lysate;
III) RT reaction without and with drug the RT-containing lysate from step II) is divided into at least two aliquots and the enzymatic polymerization process is carried out in parallel in the absence of the selected drug and in the presence of a predetermined concentration of the selected drug;
IV) conjugate binding: adding an antibody-enzyme conjugate solution for binding the conjugate to the polymeric DNA strands obtained from step III);
v) substrate reaction: adding a luminescent substrate of the conjugated enzyme for quantifying the enzyme activity;
VI) evaluation: for each of the two aliquots (in the presence and absence of drug, respectively), converting the light signal from the substrate into the activity of the RT present in the lysate;
and
VII) report: for each patient sample, the viral load (VL outcome) and HIV resistance (HDR) were reported, wherein the value obtained for the aliquot in the absence of drug was correlated to the amount of virus in the patient (VL outcome), and the ratio between the outcomes in the presence and absence of drug gave the RT residual activity, which is a measure of the level of drug susceptibility of the virus that had infected the respective patient.
Individual steps will now be further described for enhancing an understanding of the method and system of the present invention.
Steps I) to II) correspond to the extraction and isolation of viral enzymes and can be carried out manually or by means of a programmable automation workstation (for example a Tecan free EVO 150 liquid handling workstation).
An exemplary protocol for isolating viral Reverse Transcriptase (RT) from a biological sample will now be described. For the purposes of illustration and not of limitation, reference is made primarily to fig. 2 to 4 of the accompanying drawings in connection with the description of the specific steps I) to II) described above.
An amount of "sample additive" was first added to the wells (fig. 2). The purpose of the sample additive is to destroy free host enzymes in the plasma while leaving enzymes contained within the virion intact. In one example, a sample additive (e.g., 5' -dithiobis- (2-nitrobenzoic acid)) is pipetted into the wells of a deep well microplate. Next, a volume of patient sample (e.g., EDTA plasma from HIV-infected individuals) is added. The sample additive is mixed with the plasma (e.g. by pipetting) and incubated at room temperature (18 ℃ to 32 ℃). The virus particles are purified from sample additives, enzyme activity blocking antibodies, antiviral drugs, and other substances present in plasma that can interfere with viral RT quantification. Such purification can be achieved by several separation methods. The protocol described here is based on the use of magnetic beads with immobilized anion exchangers (e.g.in citrate buffer)
Carefully mixing magnetic beads (b)
The beads were then washed as illustrated in fig. 3. An aliquot of the bead wash solution was added to each well on the plate. The deep well plate was placed on a shaker, after which it was moved to a magnet holder and magnetized. The bead wash buffer and waste can now be removed/drained by aspiration. The washing step removes enzyme activity blocking antibodies, antiviral drugs, and other substances that may interfere with viral RT quantification. The virus-immobilized beads are now suspended in a substantially pure bead wash solution. This buffer is used for virus purification but is not applicable to the conditions required for enzymatic reaction of retroviral RT. In subsequent washes, the bead wash buffer was changed to bead conditioning buffer. The bead conditioning buffer contains recombinant proteins a-G to eliminate the remaining RT inhibitory antibodies. The virus-immobilized beads are now in RT reaction-compatible buffer.
Next, an extraction of the enzymatic activity RT will be performed, which is schematically shown in fig. 4. Lysis buffer was added to the wells. In one example, such a lysis buffer may correspond to a lysis buffer comprising a viral lysis detergent (e.g., 1.0% SynperonicA 11)TM) RT assay compatible buffer (b).
The deep well plate is then incubated for several minutes, after which it is moved to a magnet rack and magnetized. The lysis buffer causes the viral envelope to rupture allowing the RT to be released into the buffer solution. After lysis, substantially pure RT (RT lysate) in lysis buffer can be aspirated from each well, and the lysed viral envelope is captured to the walls of the well by magnetic beads. The recovered RT lysates are essentially free of RT blocking antibodies, antiviral drugs and cellular polymerase activity and can be characterized and quantified using a sensitive RT activity assay (e.g., the Cavidi ExaVir RT assay disclosed in WO 01/01129).
In step III), the level of enzymatic activity (e.g. RT activity) in the recovered lysate is determined, which is referred to as "enzymatic reaction step", or in the case of HIV viral enzymes, as "RT reaction step". The RT reaction step may be performed, for example, by using a modification of the Cavidi RT assay (disclosed in WO 01/01129). According to this protocol, poly (rA) (prA) covalently bound to wells of a microtiter plate (e.g., 96-well plate) is used as a template for incorporation of 5-bromodeoxyuridine 5' -triphosphate (BrdUTP) during the reverse transcription step at 37 ℃. This is schematically shown in fig. 5.
In steps IV) to V), and as shown in fig. 6 to 7, the amount of bromodeoxyuridine monophosphate (BrdUMP) incorporated into DNA is then detected with an alkaline phosphatase (alkaminesphatase, Ap) -conjugated anti-BrdU monoclonal antibody. There are several commercially available Ap substrates available that provide varying levels of detection sensitivity (e.g., disodium p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate) and). The latter is based on chemiluminescence and is one of the most sensitive Ap activity detection systems currently available.
According to the invention, prior to the RT reaction step, each RT lysate is divided into at least two aliquots: a first aliquot and a second aliquot. During the subsequent RT reaction step (described above), the enzyme activity in the first aliquot is measured in the absence of any drug and the enzyme activity in the second aliquot is measured in the presence of a predetermined concentration of an antiretroviral drug. The measurements are performed in parallel, i.e. simultaneously.
Evaluation (step VI)) involves measuring RT activity in the absence of drug (viral load) and in the presence of drug (drug sensitivity), respectively, and the results of such evaluation can be summarized and provided to the user as a report, which is shown in the table of fig. 1B, step VII). The table of step VII) is considered as an example and provides the results of said prior evaluations as to the viral load and drug sensitivity of the virus that has infected the patient. The table herein reports that
The evaluation step VI) of the assay according to the invention will now be described further below.
The RT reaction procedure performed on aliquots in the absence of drug yielded results on the "viral load" in patients: that is, the activity of RT recovered in the lysate is correlated with the amount of virions per volume of plasma. Here, the output (step VII)) from such a "viral load assay" (VL assay) reflects the concentration of HIV virus in the original patient sample.
The RT reaction protocol (i.e.the resistance indicator assay) performed on aliquots in the presence of antiretroviral drugs is complementary to the VL assay. The results of the drug resistance indicator assay reflect whether the HIV virus recovered from the plasma sample is resistant to drug treatment. Several antiretroviral drugs act by preventing viral replication, for example by blocking the activity of reverse transcriptase. The effect of antiretroviral drugs on reverse transcription can be measured in the RT reaction step of the viral load assay. As understood from the foregoing, the RT in the lysate is derived from HIV particles in the biological sample. If HIV in a patient develops resistance to a drug, the RT in the lysate will then show resistance to the drug in the RT reaction step. Incorporation of BrdUTP in the presence of drug will be less affected and more affected if RT is drug sensitive than a standard RT reaction in the absence of drug.
In order to quantify resistance and make comparisons between samples, the level of drug sensitivity has traditionally been expressed as an IC50Units of value (inhibitory concentration 50). This is the drug concentration that inhibits viral growth or enzymatic reactions in cell culture to 50%.
IC from drug sensitivity assays50The values may be used to make clinical decisions. IC exceeding a certain thresholds0The values indicate that the drug in the patient's antiretroviral drug therapy is not acting and that the treatment switch should be considered (pirnti et al 2017).
According to the prior art, typical ICs50Titration was performed by generating an inhibition curve in which residual RT activity was measured in the presence of serial dilutions of the drug.
This is illustrated in fig. 8A. Recombinant HIV RT enzymes with the indicated amino acid substitutions in the HIV 1BH10 sequence were incubated in RT reaction solution for 3 hours without drug and in the presence of the indicated concentrations of EFV. The remaining activity at each drug concentration was calculated as the ratio of RT activity in the presence of drug to RT activity in the absence of drug (quota) multiplied by 100 and plotted against drug concentration in μ M. As shown herein, at increased drug concentrations, the remaining activity of all tested reverse transcriptase variants decreased, but some RT variants were less sensitive to EFV and others were more sensitive to EFV. IC measured for the enzyme analyzed50Values are indicated by arrows.
According to the present invention, the evaluation is performed by an "indicator assay" in which one single drug concentration is selected for testing the effect on the remaining RT activity, also referred to herein as "single point measurement". This is illustrated in fig. 8B and 8C, which highlight the remaining RT activity at 10 μ M EFV concentration for each test sample. The results from the indicator assay are reported, for example, as the residual activity (Ra) after RT reaction in the presence of a predetermined concentration of drug.
The use of a single drug concentration provides a number of advantages. It is possible to rank the samples tested in order from drug sensitive (e.g. wild type RT) to drug resistant (e.g. mutant rRT with known sensitivity). Furthermore, only two assays are required per sample: one is activity without drug and one is activity at a single drug concentration. The choice of the fixed, predetermined drug concentration depends on the drug to be tested.
In one embodiment, the drug concentration is set to correspond to the IC for a reference RT with known sensitivity50Concentrations, e.g. IC, against mutant recombinant RT with a certain sensitivity50The value is obtained. Drug concentrations and resistance reference RT can be selected to represent clinically significant cut-offs, such as cut-offs where drug treatment apparently fails. The sample can then be scored as at least the same resistance as the reference or less resistant. Even for accurate IC of the sample50The value is unknown and the results can still be used to support therapy switch decisions.
A system according to the invention is schematically shown in fig. 15. The system (100) comprises a computer (110) and a device (112) for determining enzyme activity in a sample, the device (112) being configured to transmit an enzyme activity value to the computer (110), and wherein the computer is configured to receive the enzyme activity value and a series of standard values that correlate enzyme activity values to viral loads. The standard value is preferably stored in a computer accessible database (114). The computer (110) is programmed to determine the viral load in the sample from the enzyme activity in the absence of the drug based on a standard value correlating enzyme activity values to viral load; and determining the resistance of the virus to treatment with the drug from the relationship between the enzyme activity in the presence of the drug and in the absence of the drug.
According to some method aspects of the invention, as shown in fig. 15B, the means (112) for determining enzyme activity may further comprise means (116) for automatically extracting viral enzyme from the sample.
The means for determining the enzymatic activity (112) and the means for automatically extracting the viral enzyme (116) can be realized by commercially available laboratory equipment, such as automated laboratory robots and workstations, magnetic bead separation racks, reaction plates, plate readers, plate washers. The computer (110) may be any computer that is programmable to perform the calculations included in the method according to the invention. The computer preferably contains output means to communicate the results to a user of the system. Such devices include, but are not limited to, displays, printers, and communication lines with other devices (e.g., other computers, databases, etc.) for presenting or storing results. Some exemplary embodiments of the system according to the present invention are provided in the following examples.
FIG. 16 is a schematic representation of one embodiment of an apparatus (112) for determining enzyme activity and an apparatus (116) for automated extraction of viral enzymes. The diagram shows the setup on a commercially available laboratory workstation (Tecan tracing AG, Switzerland). The workstation contains reagent frame (1), sample frame (2), pipettor pipette frame (3), pipettor waste tip collector (4), pipettor waste liquid collector (5), magnet (6), board heater (7), shaking table (8), incubate the storage position (9) of lid, buffer frame (10), incubate lid (11), board scrubber (12), luminometer (13), cleaning solution container (14), scrubber waste liquid container (15) and board washing buffer heater (16).
Examples
Material
Separating the beads:
Magnetic bead separation plate: alpaqua Magnum FLX
An automated workstation: tecan
Chemiluminescent AP substrate:
Luminous board reader:
Hydroflex plate washer. Microplate washer available from Techan.
RT reaction plate: microtiter plates with immobilized prA, i.e.Nunc coated with prATMNucleoLinkTMLath (ThermoFisher).
Cysteine modifier: for example 66mM of 5, 5' -dithiobis- (2-nitrobenzoic acid),
mild thiol reducing agent: 33mM cysteamine in water
Protein A/G: recombinant protein A/G fusion protein combining IgG binding domains of both protein A and protein G, ProSpec-Tany Technogene Ltd. Rehovot Branch 179Herzel St. Rehovot 76110, Israel
Antiviral drugs: nevirapine (11-cyclopropyl-5, 11-dihydro-4-methyl-6H-bipyridino [3, 2-b: 2 ', 3' -f)][1，4]Diaza derivatives-6-one) (NVP), efavirenz ((-) 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1, 4-dihydro-2H-3, 1-benzo
3 '-azido-3' -deoxythymidine triphosphate (AZT-TP) was purchased from Moravek Biochemicals, California US.
Plasma samples from HIV-infected individuals: plasma samples from HIV-infected South African Blood donors were purchased from South African National Blood Service (SANBS), Biorepository, Hospital Road, Boksburg.
Recombinant RT enzyme: RT with NNRTI-specific mutations. Recombinant RT with C-terminal His-tag _ pET-30a (+) was constructed by introducing mutations into specific positions from HIV BH10 isolated beads used as WT RT sequences. Plasmid constructs were purchased from Genescript. Each rRT was expressed from a plasmid with the desired sequence, rather than by site-directed mutagenesis of the WT BH10 sequence. Plasmid DNA constructs with HIV-RT wt, L100I, K103N/L100I and Y181C were transformed into BL21(DE 3). RT protein was purified on a 4ml Ni2+ -agarose column.
RT with AZT-specific mutations was generated by introducing mutations into the RT coding region of the wild type HXB2-D EcoRI-NdeI restriction enzyme fragment cloned into the expression vector pKK233-2(Amersham Biotech). Mutations were generated using QuikChange (Stratagene). The mutant vector was transformed into E.coli (E.coli) strain XL1-Blue and the genotype was verified by DNA sequence analysis.
Recombinantly expressed and purified SIV RT was derived from molecular clone pSIVsmand/H4. An expression vector pSRT. ET11c was constructed and expression of SIV-RT in E.coli strain BL21(DE3) was carried out in a common LB medium. The enzyme was purified in three chromatographic steps on a heparin sepharose CL-6B column, an agarose FF column and a phenyl sepharose CL-4B column.
Mouse Mammary Tumor Virus (MMTV) recombinant RT was a donated by the University of Telvev (A Hizi Tel Aviv University). the MMTV RT gene was derived from a pUCl002 proviral plasmid produced by the MMTV BR6 strain, expressed in E.coli DH5 α and purified from bacterial extracts (Taube et al, 1998).
Recombinant MMuLV RT prepared according to Roth et al (1985) was purchased from Pharmacia (Cat. No. 27-0925).
Virus preparation: cell culture supernatants containing FIV Virus FIV-M2 clade B clarified from cell debris by centrifugation were licensed for admission to Donatella Matteucci, Dr.A.Regrovirus Centre and virology section, Dept.of Biomedicine, University of Pisa, Italy (Matteucci et al, 1995). The obtained supernatant was aliquoted and stored at-70 ℃ until use.
Cell culture derived human T-
Bovine Leukemia Virus (BLV) derived from FLK-BLV cells is licensed for admission to the Mongolian MalikMerza doctor (Svanova AB, Uppsala Sweden). (the cell line is chronically infected with BLV). The virus culture supernatant was clarified from the cell debris by centrifugation, aliquoted and stored at-70 ℃ until use.
The UK strain of Sheep lung adenoma Retrovirus (Jaagsiekte sheath retroviruses, JSRV) is licensed for Massimo Palmarini, moredden Research Institute, 408Gilmerton Road, Edinburgh EH177JH, UK. The obtained supernatant was aliquoted and stored at-70 ℃ until use.
PERV from PK15 cells was licensed for use by Jonas Blmberg, Section of clinical microbiology, Department of Medical Sciences, Uppsala University, Sweden. The virus culture supernatant was clarified from the cell debris by centrifugation, aliquoted and stored at-70 ℃ until use.
Buffers used:
bead wash buffer: 0.2M citrate/citric acid, 0.2M NaCl, 0.1% Triton X305, pH 6.0.
Bead repair buffer: RT assay compatible buffers, e.g.10 mM Hepes (N- (2-hydroxyethylpiperazine-N' - (2-ethanesulfonic acid)), 6.25mM KAc, 50mM MgCl2*
Lysis buffer: an RT assay compatible buffer containing a viral lysis detergent (e.g. 1.0% Synperonic a11) and having the same concentration of the same components in the conditioning buffer. When treating viruses with RT sensitive to SH oxidation/modification, a thiol reducing agent, 2mM cysteamine, is optionally added.
RT reaction solution: for example 10mM Hepes pH 7.6, 19. mu.M BrdUTP, 80ng/ml odT22、4mM MgCl22mM spermine, Synperonic A110.5% (v/v), EGTA 0.2mM and BSA 0.5mg/ml, GTP.
Drug reaction solution: the same buffer as the RT reaction solution described above to which a defined concentration of antiviral drug was added.
NRTI RT reaction buffer was supplemented with 6mM ATP, pH adjusted to 7.0 and final BrdUTP concentration reduced to 1.5. mu.M.
NRTI drug RT reaction buffer was supplemented with 0.70. mu.M AZT-TP.
All buffers used for analysis of gamma-retroviral RT were supplemented with 6mM Mn2+But otherwise the same as the above buffer.
Plate wash buffer: 3mM boric acid, 0.75% Triton X-100, 0.005% (W/V) dextran sulfate, 0.2% ETOH and 0.025% NaN3。
Viral abbreviations
HIV Human immunodeficiency virus (Human immunodeficiency virus)
SIV Simian immunodeficiency virus (Simian immunodeficiency virus)
FIV Feline immunodeficiency Virus (Feline immunodeficiency virus)
JSRV sheep pulmonary adenoma retrovirus
MMTV mouse mammary tumor virus
BLV bovine leukemia Virus
HTLV Human T lymphotropic virus (Human T-lymphotropic virus)
PERV Porcine endogenous retrovirus (Port endogenous retroviruses)
MMuLV Moloney Murine leukemia virus (Moloney Murine leukemia virus)
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