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Reduction of cccDNA with long-term NUCs

Reduction of Covalently Closed Circular DNA with Long-term Nucleos(t)ide Analogue Treatment in Chronic Hepatitis B
Ching-Lung Laia,correspondencePress enter key for correspondence informationemailPress enter key to Email the author
, Danny Wonga,correspondencePress enter key for correspondence informationemailPress enter key to Email the author
, Philip Ip
, Malgorzata Kopaniszen
, Wai-Kay Seto
, James Fung
, Fung-Yu Huang
, Brian Lee
, Giuseppe Cullaro
, Chun Kong Chong
, Ringo Wu
, Charles Cheng
, John Yuen
, Vincent Ngai
, Man-Fung Yuen

DOI: http://dx.doi.org/10.1016/j.jhep.2016.08.022

Background and aims

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), a minichromosome essential for HBV replication, is supposed to be resistant to nucleos(t)ide analogue treatment. We investigated the effect of long-term nucleos(t)ide analogue treatment on cccDNA.

Among 129 patients who had been enrolled in previous international nucleos(t)ide analogue clinical trials and had liver biopsies at baseline and one year after treatment, we recruited 43 patients on long-term continuous treatment for 72 to 145 months for a third liver biopsy. Serum HBV DNA, hepatitis B surface antigen (HBsAg) levels, total intrahepatic HBV DNA (ihHBV DNA), cccDNA, HBV pregenomic RNA (pgRNA) as well as histologic changes were examined.

At the time of the third biopsy, serum HBV DNA levels were undetectable in all but one patient. The median levels of HBsAg, ihHBV DNA, and cccDNA were 2.88 logIU/mL, 0.03 copies/cell, and 0.01 copies/cell, respectively. Compared to baseline levels, there was reduction of HBsAg levels by 0.54 log (71.46%), ihHBV DNA levels by 2.81 log (99.84%), and cccDNA levels by 2.94 log (99.89%), with 49% having cccDNA levels below the detection limit. One patient had undetectable HBsAg. The median pgRNA level, measured only in the third biopsy, was 0.021 copies/cell, with 40% of patients having undetectable pgRNA.

Long-term nucleos(t)ide analogue treatment induced marked depletion of cccDNA in the majority of patients while serum HBsAg levels, though reduced, were detectable in all but one patient. Whether cccDNA depletion is sustained and associated with better patient outcome requires further study.
Lay Summary

It is generally presumed that a form of hepatitis B virus DNA, called covalently closed circular DNA (cccDNA), which hides inside the nuclei of liver cells of patients with chronic hepatitis B, cannot be reduced by antiviral treatment. The present study showed that with prolonged treatment (median period 126 months), cccDNA can be markedly reduced, with 49% of liver biopsies having undetectable cccDNA. This suggests that viral replication capacity would be very low after prolonged antiviral treatment.
10 Responses
Avatar universal
very interesting

do we have any data about nucs used?

126 weeks would be about 10years (145weeks 12years), so i guess that by 15 to 20 years of use we might see a good number of cccdna below limit of detection and undetactable to very low hbsag
very interesting, my mother and uncles are reaching the 10 year threshold, but they are in there 60s. I cant even persuade them to get a fibroscan =/ . They would be the perfect candidates to test for cccdna depletion !
Avatar universal
Interesting result of long term antivirals effect. If nucs can be safely combined with a core inhibitor to synergistically reduce virion and mature core production this might lead to an even deeper reduction of remnant infection.
unfortunately the hbsag production from integrated hbv segments is not affected by this, thus the surface antigen still might not convert in the majority of patients.
by the time those drugs will also come.  Till then we have to keep a check by nucs only and when the new drugs arrive there might not be a hard task to eliminate the virus.
well we have to think about peginterferon add on in this context, it will sure speed up hbsag conversion when used after 10years of nucs
so what Replicor is testing, nucs plus repac plus peginf seems to be a good way ?
why those patients from the article did not seroconvert even when ihHBV DNA and cccDNA was reduced by more than 99% ?
They must have enough remnant hbsag production, likely substantially from chromosomally integrated hbv dna to prevent a visible hbsag antibody.
so even such low amount of ihHBV DNA may produce enough HBsAg to cover existing HBsAb ?
studyforhope are you still on this forum? Could you kindly review the below question?

Would it be of any benefit for everyone with Hepatitis B to be on antivirals to prevent continued hbv integration into the host geonome and chromosomes, and also to prevent more mutations (core / precore ). Or is there benefit of being off treatment under immune pressure?
Avatar universal
WOW! Very nice study.. gives a positive hope to patients on nucs. Earlier i thought nucs mainly reduces dna but it acts on all parameters. Thumbs up!
Avatar universal
Wish a good luck to those close to the 145 months! 23 months in here.
Avatar universal
Thank you,studyforhope for the reminder that another source of HBsAg, other than from cccDNA, is from integrated hbvdna. This raises several questions:
1. Does integrated hbvdna survive cell mitosis? In liver cell turnover, one liver cell dies, balanced by the mitosis of another cell, I believe.
2. Lately, a lot of studies into using HBcrAg (core related) as a surrogate marker for cccDNA. So it would be interesting to measure the serum HBcrAg of very long term NUC patients. Or  may it also be affected by hbvdna integration?

I also notice there is some revival in interest in HBsAg monoclonal antibody. I believe the newer monoclonal antibodies are more specific and can form immunocomplexes with smaller footprint. May this overcome the kidney problem? Because of its ability to enter liver cells to neutralize any HBsAg, it is time to re-visit using HBsAg monoclonal antibodies to treat HBV?
When a cell with integrated hbv dna segments undergoes mitosis, both daughter cell will have the integrated hbv. This way unfortunately the portion of integrated cells will actually increase.

if a cell with the integration dies, it can be replaced by division of a non integrated or integrated cell. Some mathematical modelling is required here to predict the long term net effect on integration status.
Integration often leads to partial transformation to a more stable phenotype and selective growth tendency, forming clones.
The treatment of hbv with anti hbsag monoclonal has been tried in the past without meaningful success. Unless the hbsag titer is tiny, it is impossible to infuse enough antibody to overcome the  complexing, consuming power of the relentless ongoing de novo hbsag production.
the Israeli two monoclonal therapy was finally tried in a phase 2 in hbv transplant patients under the trade name HEPEX B.  The fda required a large number of patients for the phase 3,  so large that the company CUBISTS  had to drop the project and I did not hear of any attempt to revive it after that.  I am curious what info you have regarding new approaches along those lines.
At any rate,  a smaller footprint for the monoclonal ab is not a desirable feature, since the possibility of kidney damage is increased. Fortunately the spherical hbsag itself is a fairly large composite antigen, that will reduce this danger.
But in a previous trial with a humanized monoclonal conducted in europe, the trial was halted because substantial kidney damage occured.
Thank you very much for the comments. The preservation of integrated hbvdna during mitosis is a disappointment.
As for mAB, the Chinese scientists recently published about their new mAB, F6E6, in Gut. In an accompanying editorial," therapeutic use against chronic hepatitis B: not all antibodies are created equal",Camille Surea wrote:
"Then, upon examination of
mAb-viral particle ICs by electron microscopy, and their size characterisation by low-speed centrifugation, it appeared that
E6F6, in contrast to other anti-HBsAg
mAbs, did not aggregate viral particles. As an explanation to the superior performance of E6F6 in HBsAg clearance, the authors speculated that the small size of
E6F6-vir al particles ICs—or lack of particles aggregation—was key to an efficient phagocytosis that would not occur with larger  ICs  formed  by  other  mAbs. 14 Perhaps future selection of mAbs withtherapeutic potential should not be based on their ability to neutralise virions at
viral entry, or their affinity for HBsAg, but rather on the nature, or position of the epitope at the surface of HBV particls, and on the ability of the related mAb to form small ICs upon binding to HBsAg. "

Hope this is feasible.
Antibodies are multimedia like igM, or normally dimers like igG, or monomers likely in this case. Monomers don't crossconnect and therefore do not aggregate when ICS are formed.

Why this should provide any relevant advantage is not clear,  typically the larger the ICS the less podocyte damage,  since they are trapped in the basement membrane before reaching the ultrasensitive podocyte layer.

Another important feature is the charge of the antibody, cationic ones are more dangerous, because they can  penetrate the electrical filter of the baement membrane. This can be somewhat modified by antibody design.

The main purpose of antibody therapy is to neutralize reinfection by trapping virions.

The b cell epitopes on the surface of virions and subviral hbsag particles are quite limited and use variations of the protruding a loop.
I would be pleasantly surprised if these researchers have to offer something therapeutically relevant.
Multimers became multimedia. ...
Avatar universal
Extract from a recent paper:

Recent advances in understanding and diagnosing hepatitis B virus infection
[version 1; referees: 2 approved]
Slim Fourati,Jean-Michel Pawlotsky1,2
National Reference Center for Viral Hepatitis B, C, and D, Department of Virology, Hôpital Henri Mondor, Université Paris-Est, Créteil, France
INSERM U955, Créteil, France

New  antiviral  approaches  that  target  various  steps  and  components of the HBV lifecycle, including cccDNA, are currently being investigated, with the hope of achieving functional cure of infection or, if possible, complete viral eradication. These approaches, which have been recently reviewed51,52, include HBV entry inhibitors, such
as Myrcludex BTM, a lipopeptide mimicking the pre-S1 domain that competes  with  HBV  particles  for  binding  to  NTCP;  cytokines  or sequence-specific   nucleases   that   damage   or   destroy   cccDNA; modulators of host cellular epigenetic-modifying enzymes, such as cytokines  or  inhibitors  of  viral  protein  function,  that  functionally
silence  cccDNA;  cholesterol-conjugated  small-interfering  RNAs or  antisense  oligonucleotides  blocking  viral  replication  and  viral protein expression; RNAse H inhibitors; capsid assembly modulators  affecting  nucleocapsid  assembly,  pgRNA  encapsidation,  and
the  nuclear  functions  of  HBV  core  protein  (cccDNA  regulation and  IFN-stimulated  gene  expression);  phosphorothioate  oligonucleotides  that  inhibit  HBsAg  release;  and  monoclonal  antibodies that decrease circulating HBsAg load51,52. These agents are at early
phases of development, and further preclinical and clinical evaluations will be needed to assess their safety and efficacy.
Avatar universal
Interesting and hopeful study, but with more than 99% or almost eradication of total hbv dna as well as cccDNA one would expect a revival of the host immune system to clear the virus and its proteins such as hbsag etirely from the system, but this doen't seem to be the case here in this study. Isn't that a bit puzzling and disappointing?
Studyforhope previously explained that a flip side to antivirals, hbvdna is reduced but also inflammation - no inflammation no immune reaction. cccDNA is invisible to the immune system except through the antigens it produces and most T-cells in a chronic infection are exhausted.
Just my understanding.
Also aren't there long term side effects from using NUCS? Cancer/kidney problems/etc? Don't really see how this kind of treatment can be practical, and it doesn't even seroconvert/cure a person.
If there is any conclusion i could make from this study, it is the fact that there is no any cure of hbv even after the viral dna is almost eradicated. This means targetting the virus directly probably is not the way to follow to get a cure, but we have to try and awaken the exhausted T-cells so that the immune system itself could take care of the virus. The recent research in finding a cure to hiv in the uk is focussing exactly on reviving T-cell response to the dormant hiv virus.
i dont understand how you can be so blind, it shows that after a decade of nucs there is almost no virus left and probably peginterferon will be able to clear on most patients after such a long time, until now they tried it on 7-8 years of nuc only with very good results it is highly probable it clears on most patient after 15years of nucs
Well, i think i'm reading the same thing as you are, so i'm not blind; the paper says almost all patients had almost cleared the hbv and cccDNA from their system after a median period of 12,5 years on nucs. But despite this outstanding reduction in viral dna, almost all the patients except one had still hbsag in their body. Now let's say these patients terminate treatment with the nucs, i guess the virus would rebound back in full strength in a matter of a few weaks, reversing the positive result of the treatment of 12 years. I see this disappointing from my view point.
Oops, wrong calculation there, i mean median 10,5 years
The paper suggests that:
"As proposed by Chevaliez et al [25], we postulate that the  relatively  slow  decline  of  HBsAg  levels  is  due
to  the  expression  of  HBsAg  from
integrated  HBV  DNA."
So the point is that even though cccDNA is almost zero, HBsAg will not be zero because of integrated hbvdna. So the paper states:
"Whether cccDNA reduction or depletion is sustainable and associated with better outcome remain to be determined "
Please remember, HBsAg is just a protein, it is not infectious, i.e., cannot make new virus, even though it may suppress the immune system.
The problem with a pos hbsag is that it swallows any antibody that might be produced.  The important function of the antibody is to largely reduce the respreading of the infection from small remnants of hbv cccDNA . Thus the production of hbsag from integrated sequences becomes a major weapon for the virus to continue its uninhibited reentry into uninfected liver cells.
Thanks for your replies, i think i need some clarification here: when the study mentions 99,84% reduction of total intra-hepatic  hbv DNA, what does it mean? Does this total include integrated hbv dna? Or is it just the total sum of cccDNA and HBV DNA available in the cytoplasm, excluding integrated hbv dna and serum hbv dna?
And what is integrated hbv dna; is it hbv dna or fragments of it that have been inserted into the human genome? Is the integrated dna capable of transcribing viral protiens such as hbsag?
The total intra hbv  hepatic dna includes cccDNA , cytoplasmic dna and integrated hbv fragments to the extent that the pcr primers used are present on the fragment.  Of course there is only minimal  extracellular or serum dna included,  since a washed liver tissue sample is lysed and tested.

Integrated hbv dna are various  fragments of the hbv genome inserted at various positions into the human chromosomal dna. If such a fragment is transcribed will depend on the transcriptional activity in the genomic environment at the insertion.
Thanks again for your time and explanation; so if the 99,8% reduction in hbv dna also includes integrated hbv dna, then there is almost no virus left that would continue to make the liver cells produce hbsag and other viral protiens. Then could we say the hbsag that still persists even after a decade of treatment with nucs is just a protien that is circulating in the blood because it could not be bound by anti-hbs? It seems the body is totally incapable of reacting to the presence of the hbsag to produce anti-hbs, that the host response towards hbsag is entirely switched off.
This might tell us that the secret in totally curing hbv infection lies on how to make the immune system react the same way as when a healthy adult is infected with hbv.
The amount of hbsag produced is thousands of times higher than the amount a typical anti hbsag antibody titer is able to bind.
Thus even a fairly high reduction of the hbsag production is still able to overcome quantitively the antibody present.  The virus is basically  tremendously overpowering  the humoral immune response, so it is free to spread it's virions without being neutralized by antibody coating the virion surface.
The most important reaction of the immune system in adult infection is a powerful t cell response, that dramatically reduces hbv presence, replication and antigen expression. The antibody seroconversion is more a late
, additional reaction that helps to finish the job and establish more permanent control of the remnant cccDNA expression.
Thanks for the clarification, i'm always learning something new and useful about hbv from you and others in this forum, which i highly appreciate.
Avatar universal
Long term active Hepatitis B also has side effect such as cirrhosis and HCC. Long term use of NUCS leading to cancer has no evidence and should not be stated without proof. Sure there are kidney issues with Tenofovir, but for most patients, after 8 years of monitoring, kidney function is stable after an initial dip. Hopefully, TAF will reduce the problem further. There are statistics that show patients can clear HBsAg on NUCS treatment, it is around 4%. Hopefully this will improve as ETV was approved only in 2005 and TDF in 2008.
Avatar universal
Bumping this old thread. Would it be of any benefit for everyone with Hepatitis B to be on antivirals to prevent continued integration into genes and chromosomes, and also to prevent more mutations. Or is there benefit of being off treatment under immune pressure?
my mom and uncles are ans still on nucs for 10 + years. . She is no longer hbsag + as of june 2018. My other uncles are still hbsag + . Coincidence or long term therapy of nucs. they would of been good candidates for research.  
Hi hepbcurepls, thanks for sharing many posts about your family. It gives a lot of hope. Especially with your uncle whos been survivng HCC, is he on a transplant list?. Have any of you gotten the HBSAG Quantitative test done? By the way I have done a lot of research on Genotype C. I think there is a flase stigma around it with HCC. Also big congrats on your mom, very happy for her.
my uncle that had no cirrhosis had cancer 2x and finally just recently got a transplant. my other uncle with cirrhosis so far has no cancer and is now on taf. So my family history currently stands like this - mother - cleared hbsag , still on etv and testing to see if she will develop antibodies. uncle 1 - liver transplant still on etv. uncle 2 - cirrohsis on taf and so far no hcc. i had a hbsag quant and my quant was 1100 when i first tested and now down to 600. hopefully it will continue decreasing. i am set to start a trial in july with taf and some kind of liquid that is suppose to knock down hbsag. from what i know so far it will be nuc + liquid for 3 months then off completely with weekly blood test. i will def keep you guys posted.
The no cirrhosis and cancer is really scary like you mentioned in previous posts. The really good thing is that your family members made it to 60+ with HBV.  Most of the studies talk about people getting HCC in their 40's and 50's and 60's. Also what is really good is that your uncle has been surviving HCC for many years. Most of articles I read on HCC is that the survival is 8 to 20 months. The transplant availability is kind of sad also, you have people who have been unlucky to get infected with a disease, majority of no fault of their own, but on the other hand you have people who abuse their bodies with alcohol and fatty liver taking up spots on the transplant list.

Very good news on the HBSAG, based on the information studyforhope provided it looks like you don't have high amounts of integrated HBVDNA. I think Genotype C might be better than the other Genotypes because they are in the immune tolerant phase for a very long time, less damage. Many people with other genotypes get to the immune clearance phase a lot sooner and thats when the constant attacks on the liver and start. I think majority of the studies have been done on Genotype C so thats why its frequently mentioned.

Please keep us posted on the liquid trial that you are doing, hope it goes well.
Avatar universal
Just want to bring up a update to a old thread. This was posted 3 years ago and since the last 3 year , my mom has cleared hbsag , hoping antibodies show up soon. Also when I first tested my hbsag quantitiy I started at 1100 iu and as of my last blood test my hbsaag quant is down to 471. I have been on nucs for 4 years now. If the 10 year rule applies my starting baseline of 1100 is about 100 percent and after 4 year im down to 471. Maybe this study posted by stephen might make a lot of sense.
it definitely makes sense i'm about 9 years on tdf and my highest hbsag when i started nucs was 7000iu and it is now 850iu/ml.to get antibodies to show up there are 2 possibilities when hbsag und: hbv vaccines for at least 1 year (not good because vaccines have many metal adjuvants to increase inflammation and response to the vaccine and this can make more harm than good), Hbig therapy (hbsab extracted from blood)
hepbcurepls or even better wait for safer drugs, maybe myrcludex
Questions for you guys:

1. Do you think everyone should be on medication to prevent more integration of HBVNDA which causes cancer? Studyforhope mentioned replication + immune activity = liver cell turnover = increase risk of cancer

2. This study shows a huge reduction in integrated HBV, is this the same integrated HBV that causes cancer?
if there is replication (hbvdna) it is best to be on tdf or etv, after 5-6years add pgintf and then keep nuc checking if hbsag goes down slowly.i thin this is the best now or if hbdna low wait for better drugs like replicor or something that achieves same results with little side effects
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