Monitoring the intrahepatic immune responses in chronic HBV patients after long-term treatment with tenofovir

Many therapies for chronic viral infections and liver diseases are expensive,
have many side effects and do not cure. To develop better treatments, knowledge
of these specific diseases is necessary. At the moment it is still indistinct
which factors play a role in clearing viral infections. To get a better
understanding, our research group investigates pathophysiological mechanisms of
chronic hepatitis C to eventually identify pathophysiological markers for
disease progression. Moreover, this research could in the future lead to better
therapies.
Currently it is estimated that more than 350 million people are chronically
infected with HBV. These people have a 20% incidence of cirrhosis of the liver
and a 100-fold increased risk of developing liver cancer. HBV causes about 1
million deaths per year worldwide. Chronic HBV infection is the result of a
complex interaction between a replicating non-cytopathogenic virus, and a
down-regulated antiviral immune response. Cells of the innate immune system,
such as natural killer (NK) cells represent the first line of defense against
viral infections. NK cells contribute to the anti-viral immune responses by
direct cytotoxicity of virus-infected cells and the production of cytokines
that can control viral replication.
We have shown that, in particular, the cytokine production by NK cells in blood
of patients with chronic HBV infection is reduced, which may lead to perturbed
antiviral responses against the virus. Because replication of HBV takes place
almost exclusively in hepatocytes in the liver, it is very important to
understand the intrahepatic immunity directed against the virus in detail.
However, despite the fact that NK cells are one of the most frequent cells
within the intrahepatic lymphocyte population their role in HBV infection is
relatively unknown.
By making use of the minimally invasive technique of fine needle aspiration
biopsies (FNAB) it has been possible to monitor liver material in a safe way
and frequent so that the local anti-viral immune responses could be studied
during the course of antiviral treatment in chronic HBV patients. In the
"Tenofovir FNAB study" this reduction in HBV viral load was obtained by
treatment with tenofovir, a highly specific and potent inhibitor of HBV
replication.
In the *tenofovir-FNAB study*, we have shown that the effects on intrahepatic
NK cells following tenofovir treatment for 48 weeks were minimal despite a
normalization of ALT and viral load decline in all patients (undetectable HBV
DNA). At week 48 after initiation of tenofovir treatment serum levels in these
patients demonstrated the absence of HBV DNA and normalization of ALT, but
relatively high HBsAg levels. Almost all patients are HBeAg-negative patients.
Since it is known that HBsAg has immunomodulatory activity, a possible
explanation of the lack of recovery of the activity of NK cells may lie at the
persistent high levels of HBsAg. A proportion of patients (up to 10 patients of
the original 20 patients) are currently, approximately 3-4 years after the end
of the clinical study, still under treatment with tenofovir or tenofovir in
combination with IFN-alpha.
Virological parameters obtained from standard diagnostics showed that in a
proportion of patients a substantial decline in serum HBsAg levels had
occurred, and in some cases HBsAg loss.
In order to get to the long-term effects of treatment in the liver, the
restoration of the immune system, and a better understanding of the importance
of HBsAg, and more insight in the disruption of immunity, it is desirable to
evaluate another FNAB and blood sample from specific patients from the previous
"tenofovir FNAB study"3-4 years after start of tenofovir treatment, and also
investigates these samples for NK cell functionality. This knowledge is
important because current clinical trials for treatment of HBV are desperately
looking for approaches to achieve successful off-treatment responses in
patients to prevent that patients have to be on lifelong antiviral medication.

Study of the phenotype and function of NK cells obtained from peripheral blood
and liver of patients who are successfully being treated for at least 3-4 years
for their chronic HBV infection with tenofovir in a flow-up study (METC nr.
2008-271).

In his study, chronic HBV patients who participated in the "Tenofovir-FNAB
study" (METC nr. 2008-271) will be asked to will be asked to donate eight tubes
of heparin blood and one FNAB for research, like they have done multiple times
in the "Tenofovir-FNAB study". In addition, there will be done a FibroScan in
order to determine the degree of liver damage.
Serum and isolated blood and liver cells will partially be investigated
directly and partially be stored with a study code for future investigations.

For each patient, extra blood and one fine-needle aspiration biopsy (FNAB) will
be collected for the assessment of intrahepatic immune responses. Using this
minimally-invasive technique of fine-needle aspiration biopsy (FNAB), it is now
possible to obtain safe and frequent liver samples to monitor local antiviral
immune responses in chronic HBV patients during antiviral therapy. The
procedure is well tolerated by patients. A large series, in which thousands of
FNABs were evaluated, describes an excellent safety profile with little
discomfort reported by the patients. Furthermore, in our clinic, we are
experienced with the collection of over hundreds of FNABs without any serious
complications to the patient. The patients are familiar with the procedure
since they have participated in the "Tenofovir FNAB-study" and did not
experience any adverse events.
Venapunction and a FNAB can cause mild pain and/or a bruise.