CN115267193B

CN115267193B - Method, system and application for judging source of HBsAg in biological sample

Info

Publication number: CN115267193B
Application number: CN202211143953.4A
Authority: CN
Inventors: 鲁凤民; 顾智强; 黄鸿鑫; 陈香梅; 姚明解
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2022-09-20
Filing date: 2022-09-20
Publication date: 2022-12-27
Anticipated expiration: 2042-09-20
Also published as: CN115267193A

Abstract

The invention discloses a method, a system and application for judging HBsAg source in a biological sample. The invention establishes an HBV DNA which is used for judging whether serum HBsAg is mainly derived from closed circular DNA (cccDNA) or integrated in host cells based on the level of the serum L-HBsAg and the proportion of the L-HBsAg in the total serum HBsAg. The method comprises a step of using the amount of L-HBsAg in a biological sample collected from the subject as a marker. By the method, the individual accurate antiviral treatment can be realized, the clinical curative effect of patients with chronic hepatitis B can be improved, the clinical cure rate can be improved, the occurrence risk of hepatocellular carcinoma can be reduced, and the method has good clinical application prospect.

Description

Method, system and application for judging source of HBsAg in biological sample

Technical Field

The invention relates to the field of biotechnology, in particular to a method, a system and application for judging an HBsAg source in a biological sample.

Background

Chronic Hepatitis B Virus (HBV) infection is a major cause of viral hepatitis, cirrhosis and primary liver cancer. The recent reports released by the world health organization show that there are about 2.96 million chronic HBV infected people worldwide. Covalently closed circular DNA (cccDNA) in the hepatocyte nucleus of chronic HBV infected persons is known to be a template for HBV replication and also a major cause of maintenance of chronic HBV infection, difficult cure of drug withdrawal recurrence, and the like. Clinically, the detection of the invasiveness of cccDNA by liver tissue puncture is limited, the serum hepatitis B surface antigen (HBsAg) level is used as a substitute serovirology index which can reflect the transcriptional activity of the cccDNA of the liver tissue, and the disappearance of the serum HBsAg is also a main evaluation index of clinical cure, and is widely used for evaluating the antiviral treatment effect of patients with chronic hepatitis B. Currently, marketed anti-hepatitis B virus drugs include two classes of nucleoside (t) analogues (NAs) and pegylated interferon (peg-IFN α). Although recent clinical studies have proposed "additive", "sequential" and other therapeutic strategies to improve the clinical cure rate of patients with chronic hepatitis B, it is still difficult to achieve effective disappearance of serum HBsAg. The reason for this is that, in addition to cccDNA, integrating HBV DNA is another source of serum HBsAg in patients with chronic hepatitis b. HBV DNA integration in liver tissues of patients with chronic HBV infection is ubiquitous, and the two types of drugs, particularly the former, have limited effect on integration-derived HBsAg. For most HBeAg negative patients, the presence of integrating HBV DNA resulted in high levels of HBsAg detectable in the sera of these patients despite the low prevalence of cccDNA pools in liver tissue and varying degrees of transcriptional repression.

For patients with HBeAg negativity to continue direct antiviral drug therapy on cccDNA transcription and viral replication processes, or to further eliminate transcriptionally active integrated HBV DNA by sequential or combined immunomodulatory drugs, a determination of the primary source of HBsAg in the patient's serum is needed. The method has important significance for realizing individualized accurate antiviral treatment, further improving the clinical curative effect of patients with chronic hepatitis B, improving the clinical cure rate and reducing the occurrence risk of hepatocellular carcinoma. However, until now, there has been no means for discriminating the source of HBsAg in serum of patients with chronic hepatitis B.

Disclosure of Invention

In order to solve the problems in the prior art, the invention innovatively establishes a method for judging whether serum HBsAg is mainly from cccDNA or integrated HBV DNA based on the serum L-HBsAg level and the ratio of L-HBsAg. Specifically, the present invention includes the following.

In a first aspect of the present invention, there is provided a method for determining the source of HBsAg in a biological sample or the active state of HBV DNA in a subject, comprising the step of using the amount of L-HBsAg in a biological sample collected from the subject as a marker.

In certain embodiments, the method for determining the source of HBsAg in a biological sample or the activity of HBV DNA in a subject according to the invention comprises the following steps:

(1) A step of obtaining an amount of L-HBsAg in a biological sample collected from the subject as a measurement value;

(2) A step of comparing the measured value with a first standard value; and

(3) When the measured value is higher than the first standard value, then determining that HBsAg in the biological sample is derived or primarily derived from cccDNA, and/or determining that cccDNA is active within the subject; or when the measurement is below the first standard value, then determining that HBsAg in the biological sample is derived or primarily derived from integrating HBV DNA, and/or determining that integrating HBV DNA is active in the subject.

In certain embodiments, the method for determining the HBsAg source in a biological sample or the active status of HBV DNA in a subject according to the present invention comprises the following steps:

(1') a step of obtaining a ratio of the amount of L-HBsAg to the total amount of HBsAg in a biological sample collected from the subject;

(2') a step of comparing the ratio with a second standard value; and

(3') when said ratio is above said second standard value, then determining that HBsAg in said biological sample is derived from or predominantly derived from cccDNA, and/or determining that cccDNA is active within said subject; or when said ratio is below said second standard value, then determining that HBsAg in said biological sample is derived or predominantly derived from integrating HBV DNA, and/or determining that integrating HBV DNA is active in said subject.

In certain embodiments, the method for determining the HBsAg source in a biological sample or the active status of HBV DNA in a subject according to the present invention comprises: a step of making a judgment in combination with the amount of L-HBsAg in a biological sample collected from the subject and the ratio thereof to the total amount of HBsAg.

In certain embodiments, the method for determining the source of HBsAg in a biological sample or the active status of HBV DNA in a subject according to the invention, wherein the ratio is further calculated by measuring the amount of L-HBsAg and the total amount of HBsAg using an agent.

In a second aspect of the invention, there is provided the use of a test agent in the manufacture of an antiviral therapy related composition, wherein the test agent comprises a first agent capable of displaying an amount of L-HBsAg.

In certain embodiments, the use according to the invention, wherein the detection agent further comprises a second agent capable of displaying the amount of HBsAg.

In certain embodiments, the use according to the present invention, wherein the antiviral therapy related composition comprises a composition for guiding antiviral personalized therapy, a composition for guiding a medication regimen, a composition for predicting the efficacy of a drug, a composition for precise antiviral therapy.

In a third aspect of the invention, a system is provided, comprising:

a data acquisition unit configured to be able to acquire data of an amount of L-HBsAg and optionally data of a ratio of the amount of L-HBsAg to a total amount of HBsAg in a biological sample collected from a subject;

a data storage unit configured to store at least data from the data acquisition unit;

the data processing unit is arranged to be communicated with the data storage unit, retrieve data in the data storage unit and compare the data with a standard value to obtain a data processing result; and

an output or display unit configured to be able to output or display the processing result.

In certain embodiments, the system according to the present invention, wherein the system is selected from at least one of a system for determining the source of HBsAg in a biological sample, a system for determining the active state of HBV DNA in a subject, a system for directing anti-HBV viral personalized therapy, a system for directing anti-HBV viral dosing regimens, a system for predicting the efficacy of anti-HBV viral drugs, a system for precision anti-HBV viral therapy, and a system for screening anti-HBV viral compounds.

In a fourth aspect of the present invention, there is provided a method of screening for a compound useful for treating or alleviating HBV infection, comprising:

a. a step of measuring the amount of L-HBsAg in the biological sample collected from the model and/or the ratio thereof to the total amount of HBsAg as a first measurement value;

b. a step of applying a test compound to the model;

c. a step of measuring the amount of L-HBsAg in a biological sample collected from the model after administration of the test compound and/or the ratio thereof to the total amount of HBsAg as a second measurement value;

d. a step of comparing the first measurement value with the second measurement value; and

e. screening a test compound as a compound useful for suppressing cccDNA transcriptional activity when the first measurement value is greater than the second measurement value, and screening the test compound as a compound useful for suppressing integrated HBV DNA transcriptional activity when the first measurement value is less than the second measurement value.

The invention detects the level of the L-HBsAg in the serum of the patient with chronic hepatitis B and calculates the ratio of the L-HBsAg in the serum, thereby having important significance and good clinical application prospect for realizing individualized accurate antiviral treatment, improving the clinical curative effect of the patient with chronic hepatitis B, improving the clinical cure rate and reducing the occurrence risk of hepatocellular carcinoma, and being helpful for further guiding the clinical antiviral treatment practice.

Drawings

FIG. 1 shows that the dslDNA expression plasmid (p-dslDNA) can mimic the expression of dslDNA-derived integrated HBV DNA fragments. (A) schematic diagram of dslDNA expression plasmid (p-dslDNA). (B) p-dslDNA plasmids were transfected into HepG2 cells (using 1.2 XHBV expression plasmid and backbone empty as control, beta-tubulin as reference protein, and intracellular HBsAg and HBcAg protein levels detected by Western blot). (C) HepG2 cells were transfected with p-dslDNA plasmid (1.2 XHBV expression plasmid and backbone empty as controls, respectively), and cell culture supernatants were collected after 72 h and HBsAg levels of each group were assayed by CLIA. (D) The same experimental procedure as (C) was performed, and the HBeAg level of each group of supernatants was measured by CLIA.

FIG. 2 shows that the level of supernatant L-HBsAg and the ratio of L-HBsAg are potential markers for discriminating the source of HBsAg. (A-B) the same copy number of prcccDNA/pCMV-Cre and dslDNA expression plasmids are transfected in HepG2 cells, cell culture supernatants are collected after 72 h, and the total HBsAg level (IU/mL) in each cell culture supernatant is detected by CLIA (A) and the L-HBsAg level (S/CO) is detected by ELISA (B). (C) The ratio of L-HBsAg to total HBsAg (L-HBsAg/total HBsAg, L-HBsAg ratio) in the culture supernatants of the two groups was calculated and compared. (D-E) prcccDNA expression plasmids and dslDNA expression plasmids are mixed according to different proportions and are co-transfected into HepG2 cells, cell culture supernatants are collected after 72 hours, and the L-HBsAg (D) and HBsAg levels in the supernatants are detected and the L-HBsAg ratio (E) is calculated. * DenotesP<0.001, bilateral one-way anova ortAnd (6) checking.

FIG. 3 shows the evaluation of the efficacy of serum L-HBsAg levels and their ratios to discriminate the source of HBsAg in chronic hepatitis B patients. (A-B) dividing the patients with the L-HBsAg-positive chronic hepatitis B into two groups according to the HBeAg state, randomly taking 200 cases, and comparing the serum L-HBsAg level and the serum L-HBsAg ratio of the two groups of patients. (C-E) dividing HBsAg, L-HBsAg positive chronic hepatitis B patient into cccDNA active transcriptome (serum HBV DNA) according to serum HBV DNA and serum HBV RNA level>10 ⁴ IU/ml, serum HBV RNA positive, n = 133) and integrated HBV DNA active transcriptome (both serum HBV DNA and serum HBV RNA negative, n = 304), ROC curve analysis was performed on serum L-HBsAg level (C), serum L-HBsAg ratio (D), and combined serum L-HBsAg level and L-HBsAg ratio (E) in both groups of population.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

Herein, in the open reading frame of the HBsAg messenger RNA, three in-frame start codons result in the production of three HBsAg subtypes: S-HBsAg, M-HBsAg and L-HBsAg (where S represents "small", M represents "medium", and L represents "large"). S-HBsAg contains four transmembrane domains and an "a" determinant of the main antigenic site of HBsAg, M-HBsAg contains, in addition to these domains, an additional amino-terminal preS2 sequence, and L-HBsAg contains both amino-terminal preS1 and preS2 sequences. In addition, the preS1 sequence in L-HBsAg contains the interaction domain required for taurocholate sodium cotransporter-dependent viral entry.

In the present invention, the term "amount" is meant to include content, level, absolute amount, relative amount, and the like.

In the present invention, the terms "reagent" and "detector" are used interchangeably to mean to include any reagent capable of displaying the amount of L-HBsAg and the total amount of HBsAg.

In the present invention, the term "cccDNA active" or "integrated HBV DNA active" is intended to indicate that cccDNA or integrated HBV DNA has transcriptional activity or expression activity.

Method for determining HBsAg source in biological sample or HBV DNA activity state in subject

The invention provides a method for judging the HBsAg source in a biological sample or the active state of HBV DNA in a subject, which comprises the steps of taking the amount of L-HBsAg in a biological sample collected from the subject as a marker, and particularly comprises the steps (1) to (3). The details will be described below.

Step (1)

Step (1) of the present invention is a step of obtaining the amount of L-HBsAg in a biological sample collected from the subject as a measurement value. Wherein the agent is any agent that can be used to indicate the amount or level of L-HBsAg or a fragment thereof to be detected. Preferably, the agent includes, but is not limited to, an antibody or antigen-binding fragment thereof. Antibodies herein specifically encompass monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, such as variable domains and other portions of antibodies that exhibit the desired biological activity.

The term "monoclonal antibody (mAb)" refers to an antibody that is highly specific and directed against a single antigenic determinant (epitope). Thus, the term "monoclonal" refers to an antibody to the same epitope and should not be construed as requiring the production of the antibody by any particular method. It is understood that monoclonal antibodies can be prepared by any technique or method known in the art; including, for example, the hybridoma method (Kohler et al, 1975, nature256: clackson et al, 1991, nature 352; and Marks et al, 1991, J.mol.biol.222.

In the present invention, an "antibody fragment" refers to a molecule that is different from an intact antibody, comprises a portion of an intact antibody, and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, fv, fab '-SH, F (ab') 2; a diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The reagent of the present invention may comprise other components in addition to the above-mentioned antibody. Examples of other ingredients include, but are not limited to, diluents, developing solutions, stop solutions, washing solutions, and the like. In certain embodiments, any one of the above substances may be separately present in a state separated from the other substances, and stored in different containers (e.g., vials), as long as they are capable of contacting each other at the time of use. In addition, preferably, any two or more of the above-described substances may be mixed to exist as a mixture.

In certain embodiments, the other ingredients may be provided in the form of a solution, for example, in the form of an aqueous solution. The concentrations or amounts of these substances, in the case of their presence in aqueous solution, are readily determinable by those skilled in the art as a function of the individual requirements. For example, for storage purposes, the concentration of the substance may be present in a higher form, and when in an operational state or in use, the concentration may be reduced to the operational concentration by, for example, diluting the higher concentration solution.

The reagent of the present invention can be further prepared as a diagnostic agent for determining the HBsAg source in a biological sample or the activity state of HBV DNA in a subject. The diagnostic agent can be in the form of a diagnostic composition, a diagnostic kit, or any other form in which a plurality of separately present reagents are used in combination.

Herein, the type of sample is not limited, and examples thereof include, but are not limited to, tissue samples or fluid samples. Tissue samples include somatic cell samples and fluid samples include blood or components thereof such as plasma, serum, and the like. The biological sample may be any sample of mammalian origin, preferably of human origin. Examples of types of biological samples that may be used in the present invention include, but are not limited to, one or more of the following: urine, feces, tears, whole blood, serum, plasma, blood components, bone marrow, cells, tissues, organs, body fluids, saliva, cheek swabs, lymph fluid, cerebrospinal fluid, lesion exudate and other fluids produced by the body. The biological sample may also be frozen, fixed, paraffin embedded or a fresh biopsy.

In the present invention, the means for measuring the amount of "L-HBsAg or a fragment thereof" can be obtained by a method known in the art. Assay methods include, but are not limited to, chemiluminescent immunoassay, enzyme-linked immunosorbent assay, immunoblotting, or the like.

Step (2)

Step (2) of the present invention is a step of comparing the measured value with a first standard value. Wherein the first standard value can be a specific value or a range of values.

In certain embodiments, the first standard value can be a blood sample test value from a normal subject. Preferably a value from a blood sample from a normal subject of an age comparable to the subject to be tested, and also preferably the first standard value and the measured value are both obtained by the same method.

In certain embodiments, the first standard value of the invention is a specific value, for example, 8.11 IU/ml. In certain embodiments, the first standard value of the invention is a range of values, e.g., 5-10 IU/ml.

Step (3)

Step (3) of the present invention is a result determination step. Specifically, when the measurement value is higher than the first standard value, then the HBsAg in the biological sample is judged to be derived from or mainly derived from cccDNA, and/or the cccDNA is judged to be active within the subject; or when the measurement is below the first standard value, then determining that HBsAg in the biological sample is derived or primarily derived from integrating HBV DNA, and/or determining that integrating HBV DNA is active in the subject.

The method of the present invention further comprises (1 ') - (3'), wherein:

step (1') is a step of obtaining a ratio of the amount of L-HBsAg to the total amount of HBsAg in a biological sample collected from the subject. The reagent is any reagent which can be used to display the total amount of the L-HBsAg or the fragment thereof and HBsAg to be detected. Preferably, the agent includes, but is not limited to, an antibody or fragment thereof.

The step (2') is a step of comparing the ratio with a second standard value. Wherein the second standard value can be a specific value or a range of values. In certain embodiments, the second standard value can be a blood sample test value from a normal subject. Preferably, the values of the blood sample from a normal subject of an age comparable to that of the subject to be tested, and also preferably, the second standard value and the ratio are obtained by the same method. In certain embodiments, the second standard value of the invention is a specific value, for example, 2.036.

Step (3') is a result determination step. Specifically, when the ratio is higher than the second standard value, then the HBsAg in the biological sample is judged to be derived from or mainly derived from cccDNA, and/or the cccDNA is judged to be active in the subject; or when the measurement is below the second standard value, then determining that HBsAg in the biological sample is derived or primarily derived from integrating HBV DNA, and/or determining that integrating HBV DNA is active in the subject.

The invention further provides a diagnostic or prognostic model for determining the activity state of HBsAg-derived or HBV DNA in a biological sample in a subject. The predictive model can be constructed by machine learning methods known in the art in combination with parameters including serum L-HBsAg levels and L-HBsAg ratios. Examples of machine learning algorithms include, but are not limited to: decision trees, a naive Bayes algorithm, a support vector machine algorithm, a random forest algorithm, regression analysis and deep learning. In a particular embodiment, the diagnostic model of the invention is derived using a binary logistic regression analysis in combination with serum L-HBsAg levels and L-HBsAg ratios as variables, which are defined as follows: +/-a + -b x [ L-HBsAg]±c×[L-HBsAg/log ₁₀ HBsAg]Wherein a, b and c are training numerical values obtained by machine learning. The values obtained for different data sets or training sets are typically different. In exemplary embodiments, a is any number from 0.01 to 100, b is any number from 0.01 to 100, and c is any number from 0.01 to 100. It is understood that the values represented by a, b, and c are only exemplary, and the values can be adjusted as needed when different sample numbers or different machine learning algorithms are used, and therefore, the adjusted values or any values outside the above-mentioned range are within the scope of the present invention.

In certain embodiments, an evaluation value is calculated based on L-HBsAg and its ratio to total HBsAg, and is determined from the evaluation value. Exemplary, evaluation value = -1.541+1.379 × [ L-HBsAg × ]]-4.585×[L-HBsAg/log ₁₀ HBsAg]And ROC curve analysis shows higher efficiency, and AUC reaches 0.87.

Method for determining the effectiveness of chronic hepatitis B treatment in a subject

The present invention further provides a method for determining the effectiveness of a treatment for chronic hepatitis b in a subject, sometimes referred to herein simply as "a method of determining the effectiveness of a treatment". As described above, the present invention determines that the amount of L-HBsAg and the ratio thereof to the total amount of HBsAg are related to the improvement of clinical efficacy, the improvement of clinical cure rate and the reduction of risk of hepatocellular carcinoma, so that the effectiveness of the treatment for chronic hepatitis B can be determined by detecting the amount of L-HBsAg and the ratio thereof to the total amount of HBsAg. Specifically, the following steps may be included:

(I) A step of measuring the amount of L-HBsAg in a blood sample collected from the subject during or after the treatment and/or its ratio to the total amount of HBsAg with an agent to obtain a measurement value;

(II) a step of comparing the measured value with a standard value, preferably, measuring the amount of L-HBsAg and/or its ratio to the total amount of HBsAg in a blood sample taken from a subject before the start of antiviral treatment or under treatment as the respective standard value; also preferably, the standard value is a value obtained from a blood sample comparable to the age of the subject, e.g. the standard values are 8.11 IU/ml and/or 2.036, respectively;

(III) when the measured value is higher than the standard value, it is determined that the treatment is not effective, and when the measured value is lower than the standard value, it is determined that the treatment is effective.

Methods for screening compounds useful for treating or slowing HBV infection

The present invention also provides a method for screening compounds useful for HBV infection, sometimes referred to herein simply as "screening method". Preferably, the method may comprise:

a. a step of measuring the amount of L-HBsAg and/or the ratio thereof to the total amount of HBsAg in a biological sample collected from the model as a first measurement value;

b. a step of applying a test compound to the model;

In the screening method of the present invention, the subject is preferably an animal model or an in vitro cell infected with HBV, for example, rat, mouse, dog, pig, monkey, chimpanzee, etc. Such animals can artificially induce HBV infection. Examples of in vitro cells include, but are not limited to, human liver cancer tissue cells, examples of which include HepG2, huh7, hep3B, SMMC-7721, MHCC97, and their cultured progeny cell lines.

Kit for detecting HBsAg source or active state of HBV DNA in subject

The present invention further provides a kit for detecting the source of HBsAg in a subject or the active state of HBV DNA in a subject, comprising reagents for measuring the amount of L-HBsAg and the total amount of HBsAg in a blood sample taken from the subject to obtain a measurement value. The reagents used in the kit of the present invention may be those described above. And will not be described in detail herein.

Examples

Unless otherwise indicated, all reagents used are commercially available.

1. Construction of dslDNA expression plasmids to mimic the expression and secretion of different components of HBsAg by integrated dslDNA

dslDNA can integrate into the genome of hepatocytes, and is considered to be the material and molecular basis for HBV integration. According to the molecular mechanism of dslDNA integration into host genome, the dslDNA (HBV 1818-3215/0-1827 nt) sequence is amplified by PCR, and comprises complete SP1, SP2 and X promoter, ENH1, ENH2 enhancer, complete preS/S Open Reading Frame (ORF) and X ORF with 3 amino acids deleted at carboxyl terminal, and then BGH polyA sequence simulating host tailing signal is inserted to construct dslDNA expression plasmid (p-dslDNA) (shown as A in figure 1).

In order to verify that the system can express HBV viral protein, p-dslDNA plasmid is transfected in HepG2 cells, 1.2 x HBV expression plasmid is used as a positive control, an unloaded framework is used as a negative control, cell culture supernatant and cell sediment are collected after 72 h, and then the amounts of HBsAg and HBeAg secreted into the supernatant and the protein levels of HBsAg and HBcAg in the cells are respectively detected by ELISA or Western blot. The results showed that the p-dslDNA plasmid expressed and secreted HBsAg (B, C in FIG. 1). However, in agreement with the integration fragment, the p-dslDNA no longer expresses HBcAg and HBeAg (C, D in FIG. 1) due to the partial deletion of the CP/BCP promoter in the dslDNA sequence and its spatial structural separation from the PreC/C open reading frame. The above results suggest that the dslDNA expression plasmid constructed in this example can mimic the expression of dslDNA-derived integrated HBV DNA fragments.

2. The level of supernatant L-HBsAg and the proportion of the L-HBsAg in the total HBsAg are potential markers for distinguishing the source of the HBsAg in serum of a patient

Since HBsAg has dual sources of cccDNA and integrated HBV DNA fragments, and the latter cannot inhibit HBsAg from integration source due to the independent of the viral replication system of cccDNA, the direct anti-HBV therapeutic drug is suitable to adjust the therapeutic measures. However, it is not clinically determined whether HBsAg in serum of a patient with chronic hepatitis B is derived from integrated HBV DNA fragments. In order to find a serological marker capable of distinguishing the main source of the HBsAg in the serum of a patient with chronic hepatitis B, the invention researches the secretion characteristics and the composition difference of the HBsAg expressed by integrating HBV DNA and cccDNA.

First, prcccDNA/pCMV-Cre (cccDNA similar to cccDNA can be generated by Cre/loxP mediated DNA excision) and dslDNA expression plasmid (p-dslDNA) with the same copy number were transfected in HepG2 cells, and total HBsAg (fig. 2A) and L-HBsAg levels in the supernatant were detected after 72 h.

The results showed that L-HBsAg levels in the supernatant of dslddna group were significantly lower than in the prcccDNA group (fig. 2B). In addition, the difference in the ratio of L-HBsAg levels (L-HBsAg/total HBsAg level) between the two groups was also compared, and the results showed that the ratio of L-HBsAg in the supernatant of the dslDNA group was also much lower than that of the prcccDNA group (FIG. 2C). The above results suggest that the level and proportion of L-HBsAg in the supernatant may have the potential to distinguish the primary source of HBsAg. In order to further verify whether the L-HBsAg level and the L-HBsAg ratio in the supernatant have the potential of distinguishing the main source of the HBsAg, the prcccDNA expression plasmid and the dslDNA expression plasmid are mixed according to different proportions and are co-transfected into HepG2 cells, after 72 hours, cell culture supernatant is collected, the L-HBsAg level and the total HBsAg level in the supernatant are detected by ELISA, and the L-HBsAg ratio is calculated. The results showed that, consistent with the above results, both the level of L-HBsAg and the ratio of L-HBsAg in the dslDNA group were significantly lower than in the prcccDNA group (lane 1 vs. lane 6,P<0.001 And the level of secreted L-HBsAg and the ratio of L-HBsAg in the supernatant increased significantly with increasing prcccDNA ratio in the mixed system (D, E in FIG. 2). The results show that the level of L-HBsAg secreted by prcccDNA and dslDNA in the supernatant and the ratio of L-HBsAg have obvious difference, and when integrated HBV DNA is mainly used in a mixed system, the level of L-HBsAg in the supernatant and the ratio of L-HBsAg are obviously reduced; while the level of L-HBsAg and the occupation of L-HBsAg in the supernatant are determined by the active expression of cccDNAThe ratio is obviously increased, and further indicates that the serum L-HBsAg level and the ratio of the L-HBsAg of the patients with chronic hepatitis B have the potential of distinguishing the main source of the serum HBsAg.

The cccDNA in the liver tissue of the HBeAg positive patient is mostly in a transcription active state, and at the moment, the serum HBsAg of the patient mainly comes from the cccDNA; while the cccDNA in the liver tissue of most HBeAg negative patients and chronic hepatitis B patients treated by NAs for a long time is in a transcription inhibition state, but high-level HBsAg still exists in serum, which indicates that the HBsAg in the serum of the HBeAg negative patients is mainly derived from integrated HBV DNA. To further explore the potential of serum L-HBsAg level and L-HBsAg ratio in chronic hepatitis B patients in judging whether serum HBsAg is mainly derived from cccDNA or integrated HBV DNA and judging transcriptional activity thereof, the present invention firstly compares the difference of serum L-HBsAg level and L-HBsAg ratio of HBeAg positive (n = 200) and HBeAg negative (n = 200) chronic hepatitis B patients who have not received antiviral treatment (the clinical characteristics of the two groups of people are shown in Table 1). The results showed that the serum L-HBsAg level and the ratio of L-HBsAg in patients who were HBeAg negative were significantly lower than in patients who were HBeAg positive (S/CO: 6.785 vs.12.43,P<0.001; the ratio of 2.188 vs, 3.257,P<0.001 (A and B in figure 3), namely when the serum HBsAg of the chronic hepatitis B patient is mainly derived from cccDNA, the serum L-HBsAg level and the L-HBsAg ratio are both higher; and when the serum HBsAg of the chronic hepatitis B patient is mainly derived from the integrated HBV DNA, the level and the ratio of the serum L-HBsAg are lower, and the further confirmation shows that the level and the ratio of the serum L-HBsAg of the chronic hepatitis B patient have the potential of being used as a serum marker for distinguishing the main source of the serum HBsAg.

Table 1 clinical characteristics of the study cohort

Data are represented in numbers or median (quartile range). Two-sided Mann-whitney U test or chi-square test.

In order to further prove the efficacy of the level of the serum L-HBsAg and the ratio of the L-HBsAg in distinguishing the main source of the serum HBsAg of patients with chronic hepatitis B, the invention bases on the HBV DNA and the HBV RNA level of the serum HBV of patients with chronic hepatitis B. Considering the invasiveness of quantitatively detecting cccDNA by liver tissue puncture, the invention accurately reflects the activity of cccDNA in liver tissue of patients by taking serum HBV DNA and RNA levels as alternative serological indexes, and accordingly classifies the included patients with chronic hepatitis B into cccDNA active transcriptome (serum HBV DNA)>10 ⁴ IU/ml, serum HBV RNA positive, N = 133) and integrated HBV DNA active transcriptome (both serum HBV DNA and serum HBV RNA below the lower limit of detection, N = 304), clinical features of both groups of population are shown in table 2. The research shows that the serum L-HBsAg level and the ratio of L-HBsAg of the chronic hepatitis B patient with cccDNA actively transcribed are obviously higher than those of the chronic hepatitis B patient with integrated HBV DNA active transcriptome (11.47 vs. 5.35,P<0.001; 2.91 vs. 1.79, P<0.001 (Table 2). And ROC curve analysis results showed that the serum L-HBsAg level, L-HBsAg ratio had good potency in discriminating whether HBsAg is mainly derived from integrated HBV DNA or cccDNA (AUC =0.80, 95% confidence interval: 0.75-0.84 AUC =0.71, 95% confidence interval: 0.66-0.76) (C, D in fig. 3), whereas a diagnostic model combining the serum L-HBsAg level and L-HBsAg ratio was obtained by binary logistic regression analysis, the ROC curve analysis results of which diagnostic model showed higher potency (AUC =0.87, 95% confidence interval: 0.83-0.92) (E in fig. 3). The cut-off value of the serum L-HBsAg level is 8.11S/CO (sensitivity is 78.9%, specificity is 70.4%), wherein both the sensitivity and the specificity are higher than 70% in the range of 8.07-9.12S/CO. The serum L-HBsAg has a cut-off value of 2.036 (sensitivity of 79.7% and specificity of 57.9%) to 2.636 (sensitivity of 59.4% and specificity of 73.7%) with good efficiency. The cut-off value of the combined serum L-HBsAg level and L-HBsAg ratio is-0.627 (sensitivity is 71.4%, specificity is 91.1%).

Table 2 clinical characteristics of the study cohort

In conclusion, the detection of the level of the serum L-HBsAg of the chronic hepatitis B patient and the calculation of the ratio of the serum L-HBsAg have important significance and good clinical application prospect in realizing individualized and accurate antiviral treatment, further improving the clinical curative effect of the chronic hepatitis B patient, improving the clinical cure rate and reducing the occurrence risk of hepatocellular carcinoma. The invention is helpful for further guiding clinical antiviral therapy practice, namely, the main source of serum HBsAg of a patient can be judged by detecting the serum L-HBsAg of a patient with positive or negative HBeAg and calculating the ratio of the serum L-HBsAg, if the serum L-HBsAg index of the patient is higher than the cut-off value, the transcription activity of cccDNA in liver tissue of the patient is prompted, and the direct antiviral therapy mainly aiming at the depletion or the inhibition of cccDNA, the transcription expression and virus replication of the cccDNA is continued to be carried out on the patient; if the serum L-HBsAg index of the patient is lower than the cut-off value, the serum HBsAg index of the patient is mainly from integrated HBV DNA with active transcription, and cccDNA in liver tissues is mostly in a depletion or transcription inhibition state, and the patient should be treated by sequential or combined immune regulation drug to eliminate the integrated HBV DNA with transcription activity.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

Claims

1. Use of an agent for detecting L-HBsAg for preparing a kit for judging the HBsAg origin in a biological sample or the transcription active state of HBV DNA in a subject, characterized in that the judging method comprises the following steps with the amount of L-HBsAg in blood or serum collected from the subject as a marker:

(1) A step of obtaining an amount of L-HBsAg in blood or serum collected from the subject as a measurement value;

(2) A step of comparing the measured value with a first standard value, wherein the first standard value is derived from a sample test value of a normal subject or a sample test value of a normal subject of an age equivalent to that of the subject to be tested; and

(3) When the measurement value is higher than the first standard value, then determining that the HBsAg in the biological sample is derived or predominantly derived from cccDNA, and/or determining that cccDNA is active within the subject; or when the measurement is below the first standard value, then determining that HBsAg in the biological sample is derived or primarily derived from integrating HBV DNA, and/or determining that integrating HBV DNA is active in the subject.

2. Use according to claim 1, characterized in that it comprises the following steps:

(1') a step of obtaining a ratio of the amount of L-HBsAg to the total amount of HBsAg in blood or serum collected from the subject;

(2') a step of comparing the ratio with a second standard value derived from the sample test value of a normal subject or the sample test value of a normal subject of an age equivalent to that of the subject to be tested; and

(3') when said ratio is higher than said second standard value, then determining that HBsAg in said biological sample is derived from or predominantly derived from cccDNA, and/or determining that cccDNA is active in said subject; or when the ratio is below the second standard value, then determining that HBsAg in the biological sample is derived or primarily derived from integrating HBV DNA, and/or determining that integrating HBV DNA is active in the subject.

3. Use according to claim 1, characterized in that it comprises: a step of judging in combination with the amount of L-HBsAg in blood or serum collected from the subject and the ratio thereof to the total amount of HBsAg.

4. Use according to claim 2 or 3, wherein the ratio is further calculated by measuring the amount of L-HBsAg and the total amount of HBsAg using reagents.

5. Use of a test agent in the preparation of a composition for determining the source of HBsAg in a biological sample or the transcriptional activity of HBV DNA in a subject by a method comprising the steps of:

(1) A step of obtaining, as a measurement value, an amount of L-HBsAg in blood or serum collected from the subject, wherein the amount of L-HBsAg is obtained using the first agent;

6. The use of claim 5, wherein the detection agent further comprises a second agent capable of displaying the amount of HBsAg, and the method comprises:

(1') a step of obtaining a ratio of an amount of L-HBsAg to a total amount of HBsAg in blood or serum collected from the subject, wherein the amount of L-HBsAg is obtained using the first agent and the total amount of HBsAg is obtained using the second agent;

(3') when said ratio is above said second standard value, then determining that HBsAg in said biological sample is derived from or predominantly derived from cccDNA, and/or determining that cccDNA is active within said subject; or when the ratio is below the second standard value, then determining that HBsAg in the biological sample is derived or primarily derived from integrating HBV DNA, and/or determining that integrating HBV DNA is active in the subject.

7. A method of screening for a compound useful for treating or ameliorating HBV infection, comprising:

a. a step of measuring the amount of L-HBsAg in blood or serum collected from the model and/or the ratio thereof to the total amount of HBsAg as a first measurement value;

b. a step of applying a test compound to the model;

c. a step of measuring the amount of L-HBsAg and/or the ratio thereof to the total amount of HBsAg in blood or serum collected from a model after administration of a test compound as a second measurement value;