Example entry: Hepatitis B cccDNA viral reservoirs - stubborn nails in the quest for a complete cure

Research Showcase 2023
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Hi all! I’m a PhD student under @ThomasTu working on a few projects revolving around cccDNA, which I’m sure you all know about. Briefly - cccDNA is a stable form of Hepatitis B Virus, which stays in liver cells and is a blueprint which the virus uses to make new virus particles. Getting rid of it is the tricky bit because current drugs (e.g. entecavir, tenofovir, lamivudine) don’t target this form of HBV, only the creation of new virus part of the life cycle.

One way which the body naturally gets rid of cccDNA is through cell division, our lab has shown that when liver cells divides, the cccDNA isn’t carried along into the new cells – somehow it’s lost in the process, resulting in two uninfected daughter cells. So then you’d think that if we can stop cells making new virus (through current therapy), and then wait for mitosis to naturally occur the liver would eventually eliminate all of the cccDNA in all liver cells, meaning a complete cure right?

Well, it’s a little bit more complicated because that’s not really what we see in animal models (humanised mice, chimpanzee, or duck models) or in patient data. Even if we wait until after cccDNA levels are below detectable levels to stop treatment, we see a return of virus levels.

So why is that?

Running theory that our lab has is that not every liver cell wants to divide, there is a rare population of cells (around 1 in 5000) which don’t, meaning that they’ll never lose their cccDNA. These cells basically act as a viral reservoir, hidden caches of virus which sit dormant until the right time (i.e. when stopping treatment!) to reinfect the liver.

Currently my work is focused on looking into different pathways which might cause formation of these “reservoir cells”, as well as potential treatment options to get rid of them. Super cool stuff (at least I think so) and lets me play with some pretty expensive toys (read: professional scientific equipment).

Thanks for reading and happy to take questions :slight_smile:

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Hi,
welcome to the community.
I have questions/comments.

  1. u said that cell division which results in two uninfected daughter cells is one way the body gets rid of the virus. Are you saying that one infected cell divides and turns into two uninfected cells? How? How can an infected cell turn into uninfected cells? In that case, why do we use antiviral drugs to suppress the replication because one infected turned into two uninfected no longer has a virus? Shouldn’t we let the infected cells to divide so that they can turn into two uninfected cells? I’m excluding the reservoir cells in this question.
    I think I am not understanding this correctly.

  2. u said that u r looking into different ways “reservoir cells” may be formed. Do reservoir cells form differently than the regular cccDNA?

Thank you.

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Hey, appreciate the warm welcome :slight_smile: In regards to your questions:

  1. How can an infected cell turn into uninfected cells?

We’re not exactly sure just yet, the research just hasn’t been done yet so anything I say in the next few paragraphs is speculation.

It may be due to cccDNA being unable to attach to mitotic spindles – long series of microtubules which guide DNA to either pole of the cell, helping evenly divide the DNA of the cell. I’ll explain.

Animals (including humans) have what we call “open” mitosis, which is where the nuclear envelope is broken down before mitosis occurs, and everything in the nucleus (including cccDNA) gets mixed up with everything else in the cells.

The human DNA (in the form of chromosomes) attaches to two or more mitotic spindles via a bundle of proteins known as a kinetochore. Think of this as glue holding the chromosome to the spindle. The spindles themselves are attached to centrosomes at either pole of the cell. cccDNA doesn’t have these kinetochores and thus can’t attach to the spindles.

The spindles then pull on the chromosomes like a Christmas cracker or wishbone - splitting them evenly in two and guiding the two identical halves to either side, where the nuclear envelope reassembles and the cell finishes dividing. As cccDNA was not part of this – it gets left behind in the cytoplasm where presumably it gets eaten by a Grue (or more likely a passing DNase).

Again, this is all purely hypothetical and research needs to be done to actually find evidence for anything I just said in the above paragraphs.

Shouldn’t we let the infected cells to divide so that they can turn into two uninfected cells?

If the infected cells (which is approximately 100% of your liver in chronic HBV) without drugs to supress viral replication, the newly uninfected cells will quickly become infected with new virus.
I think you may have mixed up the fact that that the drugs supress viral replication, not hepatocyte division.

  1. Do reservoir cells form differently than the regular cccDNA?

Currently, we don’t think that the cccDNA in reservoir cells is any different than the cccDNA in any other infected cell, more likely is the fact that hepatocytes are very diverse and some are more or less inclined to actually want to undergo mitosis.

However, there may be some stressors which might cause cells to stop wanting to undergo mitosis – specifically linked to inflammation of the liver. As this is an ongoing area of research, I’m afraid we don’t know too much more about this at the moment, but watch this space!

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Thank you for the thorough explanation. I’ll have to study a little bit more to understand completely. Your explanation seems to be more than sufficient. Unfortunately, I’m not familiar with bio mechanisms but something to learn about. And you are right, I did get mixed up with viral replications and hepatocyte division. From what I’ve read, HBV is much trickier than HCV so it might take longer to find a solution but glad to know that we have people like Thomas and you on our side!

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Hello henrikzhang,
Thanks for spending the time to explain this in easy to understand form. I’m not in medical field but this inspired me to comment and ask some questions as well:

  1. As per my understanding, this cccDNA doesn’t change the function of infected hepatocytes, and that innate immunity will recognize these cells and start attacking them, leaking enzymes like AST and ALT to the blood, repeated attach and recovery cycles increase the risk of fibrosis and cirrhosis after.
    if this is the case then in theory even if some cells doesn’t divide, it will be recognized and attacked, in case if antiviral drug is used for long enough (years or even decades), there will be a probability that the virus is completely cleared at some point. am I right?

  2. There is a latent phase of the infection where the virus is undetectable in blood as a result of natural immunity or using treatment, does it really mean that the virus is latent, or it’s still active in hepatocytes producing copies nonstop but these copies are either neutralized by antibodies or antiviral drug?

Thank you again for your effort.

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Hi @Moe,

  1. You’re on the right track, and in some cases yes there is a complete clearance of the virus and antiviral treatment can be stopped. However, we do currently theorise that infected reservoir cells may be able to avoid being targeted by the immune system, which could either be due to the host cell itself releasing various immune-supressing compounds, or possibly due to HBV itself reducing its production of new virions and HBsAg in order to ‘lie low’. Currently we’re looking into the possibility of the former, however it wouldn’t surprise me if there were elements of the latter which contribute as well, given that other viruses have been known to do so (mainly those in the herpes family, which are responsible for such diseases such as cold sores and chicken pox).

  2. By latent stage, I believe you’re referring to the immune-control phase, or as it’s now supposed to be called “HBeAg-negative chronic HBV infection”. I think we’ve moved away from calling it latent because of exactly that, it’s not really latent – while you see loss of HBeAg and overall decrease in HBsAg and HBV DNA, the virus is still active in the liver, or as much as it can be if its replication is suppressed by antiviral drugs.

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Dear Moe,

A few minor course corrections here…

In hepatocytes with active cccDNA (and integrated HBV DNA), the lipid recycling and secretory pathways are hijacked by the production of subviral particles (which are biochemically very similar to HDL). Additionally, the gene expression and metabolic function of HBV infected cells is altered versus infected cells. As such hepatocytes containing active cccDNA or integrated HBV DNA will have altered metabolic function and do not contribute to normal liver function. This is why host (immune mediated) transaminase flares in chronic HBV infection can be very high (> 3000 U/L) and persist for more than 6 months without any symptoms or impact on liver function. Infected cells are no longer part of the reservoir of normal liver function and their removal does not impact liver function OR lead to increased fibrosis or cirrhosis. In fact, host mediated transaminase flares are typically associated with improved liver disease, improved virlogic status and functional cure.

Transaminase flares are caused primarily by T-cell mediated clearance of infected hepatocytes, not innate immunity. There is a cytolytic aspect to innate immunity (these are called natural killer or NK cells). However, NK cells cannot discriminate between infected and uninfected cells. The activation of these cells is typically the cause of dramatic alteration of liver function seen in acute HBV infection or HBV flares during the natural course of infection.

The progression of fibrosis is driven by long term liver inflammation which is a function of innate immunity. This is one of the challenges of diagnosing HBV infection - the production of subviral particles suppresses innate immunity while viral replication proceeds. Often people are finally diagnosed with HBV infection becuase they don’t feel well or have signs of liver disease only after many years of HBV infection.

It is important to be aware that cccDNA is decorated with the same proteins that your chromosomal DNA is decorated with. Thus cccDNA is “chromatinized” and is more correctly thought of as a “HBV minichromosome”. In cells with inactive cccDNA, no viral antigens are produced so these cells cannot be recognized by either innate or adpative (T-cell) immunity. Most of this latent cccDNA is in a highly condensed form called heterochromatic cccDNA (most of your cellular DNA is also in this form) which cannot be targeted by any antiviral approach we are aware of. While there is good data to show that all forms of cccDNA can be diluted out by cell division over a long period of time, there always remains a persistent reservoir of latent cccDNA which, regardless of how small it becomes, can reactivate HBV infection with loss of innate immune control. This is a phenomena which has been described in patients taking immunosuppressive therapy who previously had no evidence of HBV infection.

We also have good clinical data that shows that even when all detectable signs of HBV infection are absent in the liver after long term therapy with approved drugs like ETV and TDF, infection rebounds very quickly from this tiny reservoir of latent cccDNA due to the inhibition of innate immune control by continued production of SVP from integrated HBV DNA. A good (but a bit technical) paper to read on this is Rebound of HBV DNA after cessation of nucleos/tide analogues in chronic hepatitis B patients with undetectable covalently closed - PubMed.

Self resolution of HBV infection following acute HBV infection (common) or functional cure of HBV with treatment of chronic HBV (rare with approved therapies) are the current definitions we use for truly latent HBV infection. In both cases, reactivation of latent cccDNA is suppressed by a normally functioning innate immune response made possible by the absence of circulating subviral particles. In the case of functional cure, removal of integrated HBV DNA is required to clear subviral particles.

Hope this helps!

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Is it speculated that the cells that tend not to divide may be senescent? In which case perhaps foxo4-dri could target them (if they do exhibit p53)

Does alcohol kill random liver cells? I wonder if virally suppressed people with very low Hbsag could just start slamming alcohol to take out random chunks of the liver until they’re gone

And one more question: given that mitosis does appear to reduce cccdna in most cases why isn’t myrcludex b more effective against HBV? Do you know if it’s been tested alongside a NUC and an immune system modulator like interferon or thymosin alpha 1?

Sorry for the hair brained ideas. They’re just lateral thought experiments, not suggestions

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Hi Bob,

Fantastic questions!

  1. This is something we’re pursing at the very moment. More news to come!
  2. Slamming alcohol is definitely something that we would recommend AGAINST. Alcohol metabolism is not random in the liver; particular liver cells are more susceptible than others to cell death. This approach would probably need an unacceptable amount of widespread liver damage, so it needs to be something specific and/or slow going over time.
  3. There isn’t very much liver turnover that occurs over time, it has been tested with interferon and the effect on HBV surface antigen levels is low.

Cheers,
Thomas

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Hi @ThomasTu

Thanks for the clarification on those points

Regarding the first point about senescence, I did take 80mg of foxo4-dri intravenously a few months ago. But I was treatment naive back then, I only started NUC’s a week ago. I just sporadically had blood tests before and afterwards and there were various fluctuations, but I don’t know what’s attributable to the foxo4-dri because I had a bad year last year with HBV flares and stuff. Anyway, if it could be of any help I’d share my bloods with you and do anything else that may be useful

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Thanks Bob,

How interesting! We may follow up with you as our research continues.

Cheers,
TT

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Regarding your research, does this help clarify the persistence of cccdna?
https://www.cmghjournal.org/article/S2352-345X(22)00219-3/fulltext

Also, it states there that "Lysosome inhibitors did not decrease HBV infection but rather increased its replication (chloroquine) ". I was curious though, in light of the above, do you think that this could have any merit considering they’re claiming a 100% success rate in curing it? https://rjptonline.org/HTMLPaper.aspx?Journal=Research%20Journal%20of%20Pharmacy%20and%20Technology;PID=2008-1-3-67

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Dear @bob,

Thanks for bringing these interesting papers up for discussion. The first mostly concerns how new cccDNA is formed, rather than how to get rid of existing cccDNA. Preventing new cccDNA formation is relatively easy and can be done with current therapies, the harder task is getting rid of cccDNA that is already there.

Regarding chloroquine, I can say that we have done this experiment and have found that it does increase virus replication in vitro. Regarding the second study, it is highly suspect. They have not included a control group, nor have they registered this as a clinical trial. All work on humans has to be cleared through an ethics approval board, and this group has not provided information on this. There are also numerous typographical errors in it.

Hope this helps,
Thomas

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Thank you for the explanation. It makes me happy to hear you’re researching this and mapping things out more and more. Happy holidays to everyone!

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