tóm tắt: Nghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBV

Nghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBVNghiên cứu mối liên quan giữa cccDNA tế bào gan với HBV DNA, HBV RNA huyết tương ở bệnh nhân viêm gan B mạn tính và xơ gan do HBV

MILITARY MEDICAL UNIVERSITY

DO THI LE QUYEN

RESEARCH ON CORRELATION BETWEEN INTRAHEPATIC cccDNA, SERUM HBV RNA AND HBV DNA IN CHRONIC HEPATITIS B AND CIRRHOTIC PATIENTS DUE TO HBV

Specialization: Infectious and tropical diseases

Code: 9720109

SUMMARY OF DOCTORAL THESIS

HA NOI – 2024

Scientific Supervisors:

1 Asc Prof PhD Hoang Tien Tuyen

2 PhD Ho Huu Tho

1st: Contradictor: Prof PhD Bui Vu Huy

2nd Contradictor: PhD Bui Tien si

3rd Contradictor: Asc Prof PhD Nguyen Dang Ton

This doctoral thesis will be defended at the University ‘s

Committee of Grading at the before the Vietnam Military MedicalUniversity at:…… on…….20…

WHO 2023 estimates that 296 million people were living withchronic hepatitis B infection in 2019, with 1.5 million new infectionseach year Hepatitis B resulted in an estimated 820 000 deaths,mostly from cirrhosis and hepatocellular carcinoma ( HCC)

cccDNA (Covalently closed circular Deoxyribonucleic acid) plays animportant role in the persistence, maintenance and replication of thevirus in liver cells, while HBV DNA does not accurately reflect theactivity of cccDNA In addition, cccDNA extraction and analysisrequires liver biopsy, an invasive technique that often does notrequire the cooperation of many patients Therefore, finding a marker

in plasma that can predict or represent cccDNA is extremelynecessary HBV pgRNA (HBV RNA) is a transcription product ofcccDNA circulating in plasma This marker has the potential to trulyreflect the activity status of cccDNA in liver cells, especially inVietnam, where it has not yet been established Is there any research

on this issue?

Thereofore, we conducted the project: "Research on therelationship between intrahepatic cccDNA and HBV DNA, HBVRNA in patients with chronic hepatitis B and cirrhosis due to HBV"with two goals

1 Determine hepatic cccDNA concentration, HBV DNA viral load and plasma HBV RNA concentration in patients with chronic hepatitis B and cirrhosis caused by HBV.

2 Assess the relationship between hepatic cccDNA concentration with HBV DNA viral load and plasma HBV RNA concentration in patients with chronic hepatitis B and cirrhosis caused

by HBV.

NEW CONTRIBUTIONS OF THE THESIS:

This thesis is the first study in Vietnam to quantify cccDNAlevels in liver cells in patients with chronic hepatitis B and cirrhosiscaused by HBV The cccDNA concentration in the two groupsHBMT and cirrhosis were 1.61 ± 0.40 and 1.56 ± 0.39log10copies/cell, respectively cccDNA is correlated with HBVRNA, HBV DNA in descending order as follows: in the same twogroups, the correlation between cccDNA and HBV RNA is r= 0.57;p<0.01), then cccDNA with (HBV DNA+RNA) is r=0.47; p<0.01and the lowest correlation coefficient is between cccDNA and HBVDNA (0.21; p= 0.01) Thus, it can be seen that HBV RNA is avaluable marker to evaluate the level of cccDNA activity in patients

with HBV infection in Chronic Hepatitis B and cirrhosis caused by HBV.

STRUCTURE OF THE THESIS

The thesis consists of 122 pages with 4 chapters, 36 tables, 9figures and 10 charts, 2-pages introduction; 34 pages overview; 26pages subjects and methods; 28 pages discussion; 2 pagesconclusion; 1 page recommendations, 1 page: limitations of thestudy, 128 references

CHAPTER 1 – OVERVIEW 1.1 Situation of HBV infection in the world and in Vietnam

1.1.1 Situation of HBV infection in the world

According to the World Health Organization (2023), there are anestimated 296 million people who are chronic carriers of hepatitis Bvirus (Hepatitis B virus: HBV) globally Chronic HBV infection isalso the cause of about 820,000 deaths each year related tocomplications of the disease such as chronic hepatitis B (CHB),cirrhosis and hepatocellular carcinoma (HCC)

1.1.2 Situation of HBV infection in Vietnam

Vietnam is one of the countries with a high HBV infection rate inthe region and suffers from severe consequences caused by HBVinfection Data from the Vietnamese Ministry of Health in 2019reported that the proportion of chronic HBV infection ranged from 6– 20% of the total population, equivalent to about 8 million people

1.2 Pathogenesis and progression of chronic HBV infection

1.2.1 Pathogenesis of disease caused by HBV

- Life cycle of HBV

During chronic HBV infection, cccDNA is used as a template

to synthesize messenger RNAs with different sizes 3.5; 2.4; 2.1 and0.7kb These RNAs are attached to their tails and move to thecytoplasm and perform their functions Once pgRNA is synthesized,

it is packaged in the nucleocapsid and translocated into the cytosol,then initiate genome replication

The responses of HBV-specific T lymphocytes and Blymphocytes are decisive factors in eliminating free virus andinfected liver cells and play a role in long-term immune control ofHBV During chronic HBV infection, the virus will hide and inhibitthe antiviral ability of the adaptive and innate immune systemsthrough different mechanisms, thereby achieving continuous,simultaneous replication The host also exhibits dysfunction ofvarious immune cells

1.2.2 Natural course of chronic HBV infection

Chronic HBV infection undergoes 5 stages: stage 1 theHBeAg-positive chronic HBV infection stage or, according toprevious terminology, the immune tolerance stage Stage 2: HBVstage with HBeAg positive, stage 3: Chronic HBV infection withHBeAg negative (corresponding to inactive viral carrier stage), stage4: HBV stage with HBeAg negative and HBsAg negative stage inwhich the disease is characterized by negative HBsAg These periodsare classified according to different characteristics based on HBeAg

carrier status, ALT elevation and ALT levels as well ashistopathological damage.

1.3 Chronic hepatitis B and cirrhosis due to HBV

1.3.1 Chronic hepatitis B virus

HBV is a condition of inflammation and necrosis of liver cellscaused by the existence of HBV lasting more than 6 months MostHBV patients have poor clinical manifestations such as only transientfatigue, sometimes loss of appetite, bloating , AST and ALTactivities do not increase or slightly increase by 2-5 times the highthreshold of the normal values

1.3.2 Cirrhosis caused by HBV

Cirrhosis caused by HBV is also known as parenchymalcirrhosis or post-necrotic cirrhosis, fibrotic lesions originate from theportal space enter the liver parenchyma to replace necrotic tissue.The liver tissue proliferates, creates large and small neoplasticnodules of irregular diameter, usually > 3mm, but mainly largenodules, so it is also called large regenerative cirrhosis

1.4.1 HBV DNA

In clinical practice, HBV DNA level plays a very importantrole in assessing viral replication, as well as prescribing andmonitoring antiviral treatment Persistent high viral load is closelyrelated to increased risk of cirrhosis and liver cancer HBV DNAlevel is considered as one of a significant criteria to access treatmentresponse

1.4.2 HBV RNA

Pregenomic RNA (pgRNA) is a direct transcription product ofthe covalent closed-circle cccDNA of hepatitis B virus, playing animportant role in the amplification and replication of the viralgenome pgRNA can be reversely transcribed into rcDNA by HBVDNA polymerase to continue a new life cycle Therefore, blockingreversely transcription will not affect the release of pgRNA intoserum Different from serum HBsAg, serum pgRNA with molecular

weight of 3.5 kb is only produced from cccDNA, therefore, it canaccurately reflect the transcription status of HBV cccDNA in livercell nuclei.

1.4.3 cccDNA

cccDNA is one of the most important components of HBV,which can exist in HBV-infected liver cells and serves as a templatefor virus replication It is a key factor in the virus life cycle andpersistent HBV infection or recurrence of hepatitis after stoppingantiviral therapy because cccDNA is very stable in nondividinghuman liver cells Furthermore, cccDNA can persist throughout theentire life cycle of liver cells, thus serves as a persistent viralreservoir

1.5 Methods for quantifying HBV RNA and cccDNA

1.5.1 Some methods to quantify HBV RNA in peripheral blood of HBV patients

Some specific techniques have been and are being applied:

- PCR/Realtime PCR technique based on the 3' RACEprinciple

- RT-qPCR technique

- Droplet digital PCR technique

- Some other techniques such as QuantiGene, IndirectQuantitative Technique

1.5.2 Methods for quantifying cccDNA

- FISH method

- qPCR method

- qPCR+ PDS method

- qPCR+ T5 exonuclease method

- Direct droplet method

1.7 Studies on cccDNA and HBV DNA, HBV RNA in patients with chronic HBV infection in the World and Vietnam

1.7.1 Research around the world

Since 1988, Hadchouel M and CS have detected HBV-RNA

in circulating mononuclear cells in peripheral blood By 1996, RNA was reported by Kock J and colleagues to be found in theplasma of patients with chronic HBV infection In 2000, Mei S D.and CS used RT-PCR technique to detect HBV-RNA circulating inthe peripheral blood of patients Author Jei L et al (2017) analyzedthe relationship between HBV DNA, cccDNA in the liver and serumHBsAg levels in HBV patients with HBV DNA below the detectionthreshold after taking oral antiviral drugs The author's resultsshowed that cccDNA and HBV DNA in the liver in HBV patientswere higher than in cirrhotic patients cccDNA had a positivecorrelation with HBsAg in the group of patients with negativeHBeAg Author Hongxing H (2018) used HBV DNA combined withRNA to reflect the activity of cccDNA in acute hepatitis B patientsand HBV patients.

HBV-1.7.2 Research in Vietnam

In Vietnam, plasma HBV-RNA is a new biomarker In 2018,author Nguyen Van Dien detected plasma HBV-RNA in 61.54% ofHBV patients with an average concentration of 6.29 ± 1.42 log10copies/mL

In 2020, author Nguyen Hong Thang and colleaguesannounced the detection of plasma HBV-RNA in 86.49% of patients

at the initial time Plasma HBV-RNA levels were higher in theHBeAg-positive group than in the HBeAg-negative group Therehave been no studies on cccDNA in patients with chronic HBVinfection in Vietnam

2.1.1 Patient selection criteria

- Patients with CHB according to the 2019 Ministry of Health

- HBsAg (+) > 6 months or HBsAg (+) and anti-HBc IgM (-).

- Histopathology: F4 follow Metavir score

- All patients have never received antiretroviral treatment

- Patients with contraindications for liver biopsy (coagulationdisorders, severe liver failure, ascites ) - Patients who do not agree

to participate in the study

2.1.3 Research time and location

- Implementation time: from December 2017 to April 2020

- Research location: Department of Infectious Diseases,Military Hospital 103 and Department of Gene and GeneticTechnology, Military Medical and Pharmaceutical Research Institute,Vietnam Military Medical University

2.2 Research Methods

2.2.1 Research design: Descriptive, prospective study.

2.2.2 Study sample size, collection time and treatment regimen

- Sample size: 135 patients

- Time of sample collection: before the patient has a liver

2.2.3 Research indicators and assessment standards

2.2.3.1 Research targets

- Age, gender, fatigue, jaundice, right lower quadrant pain

- Plasma HBV-DNA concentration

- Plasma HBV-RNA concentration

- intrahepatic cccDN concentration

- HBeAg, platelets, HAI score, fibrosis

- AST, ALT and total Bilirubin

2.2.3.2 Grouping research indicators

- Age, gender, enzymes: AST, ALT, Bilirubin

- Platelets: <140, ≥ 140; HBeAg: Positive/negative

- HAI histology index scale, Fibrosis

- cccDNA: log10copies/cell, HBV DNA: < 5, ≥5log10copies/mL;

- HBVRNA: Negative (<2log10copies/ml), Positive (≥2log10copies/ml)

- HBV RNA + DNA: Total (HBV RNA + HBV DNA)log10copies/ml)

2.2.4 Engineering principles and processes

2.2.4.1 Procedure for quantifying plasma HBV RNA levels

- Process principle: Based on one-step realtime PCR technique,with a primer/probe set that pairs with the S gene region of HBV,specially designed to help select the newly created cDNA sequence fromHBV- pgRNA, which does not pair and misamplifies HBV-rcDNA

- HBV-pgRNA positive control sample: artificially synthesizedfrom the published HBV subtype B sequence under codeMF674382.1

- Detection threshold: 2 log10 copies/mL The technicalspecificity of the procedure is 100%

2.2.4.2 Principle of quantitative testing for plasma HBV DNAconcentration:

- Based on realtime PCR Taqman probe technique, using HBVReal-TM Quant Dx kit (Sacace Biotachnologies Srl, Como, Italy), onSaCycler-96™ system, Sacace, Italy

- Linear range from 60 – 108copies/mL; The quantitativethreshold of the test is 60copies/mL

2.2.4.3 Test principle to quantify HBV cccDNA concentration in livercells

Our procedure for quantifying cccDNA in the liver is performedaccording to the method published in 2011 by author Zhong

The amplification process consists of two steps as follows (1)10μl of PSAD-treated DNA was combined with primers at al of PSAD-treated DNA was combined with primers at aconcentration of 0.5μl of PSAD-treated DNA was combined with primers at amol/l each and 1μl of PSAD-treated DNA was combined with primers at al of reaction buffer

Quantification of HBV cccDNA in liver cells by quantitative time PCR method: Using the product after the RCA process as atemplate, HBV cccDNA is further amplified a second time andquantified by real-time PCR with designed primer pairs cccDNA-specific (PCR product nucleotide positions: 1523-1870) and anadditional Taqman probe with a sequence region located betweentwo direct repeats, DR1 (at nucleotide position 1826) and DR2 (atposition 1826) nucleotide position 1592), belonging to the HBVvirus gene

real-2.2.5 Data analysis method

- Use MedCalc software version 20.019 (MedCalc SoftwareLtd, Ostend, Belgium) for statistical analysis Statistics by number,percentage, median - quartiles

- ROC curve analysis estimates predictive value Multivariablelogistic regression analysis verifies independent effects

2.2.6 Research ethics issues

The PhD student's thesis uses part of the data from the project code01C-0808-2017-3, which has been approved by the Ethics Council inBiomedical Research of the Military Medical Academy (according to

decision No 780/ Decision-HVQY dated March 28, 2018).

CHAPTER 3 - RESULTS 3.1 General characteristics of research subjects

3.1.2 Characteristics of paraclinical indicators

Characteristics of HBeAg in the two groups of CHB andcirrhosis did not differ

3.2 Intrahepatic cccDNA concentration, HBV DNA load, serum HBV RNA concentration in studied patients

3.2.1 Intrahepatic cccDNA concentration

Figure 3.1 Intrahepatic cccDNA concentration in CHB and Cirrhosis

groups are equal

Table 3.9 Relationship between liver cell cccDNA concentration

and research criteria in the CHB group

characteristic

cccDNA (log 10 copies/tb)

3.2.2 Serum HBV RNA levels in two groups of studied patients

Table 3.13 Serum HBV RNA levels in two groups of studied patients

characteristic

CHB (n=105) n(%)

Cirrhosis (n=30) n(%)

P

HBV RNA

(log10copies/ml)

X ± SD (Min - max)

4,88 ± 1,65(1,99-8,71)

4,44 ±1,38(1,99-7,91) 0,18*Negative

(n=16) 13 (12,4) 03 (10,0)Positive

(n=119) 92 (87,6) 27 (90,0)

HBV RNA levels in the CHB group were higher than thecirrhosis group, but the difference was not statistically significant