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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.
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