Aa
NUC + Interferon - What is needed to secure a cure?
I attach 3 poster abstracts from AASLD2014 regarding various effects of NUC + PegIFN therapy. I wonder what is needed to improve the response and even cure rates?
1708
Functional innate immune responses are restored with sequential NUC therapy following Pegylated Interferon-Alpha exposure and not with NUC monotherapy in Chronic Hepatitis B
Upkar S. Gill1, Dimitra Peppa2, Harsimran D. Singh2, Lorenzo Micco2, Graham R. Foster1, Mala K. Maini2, Patrick T. Kennedy1;
1Hepatology Unit, Centre for Digestive Diseases, Blizard Institute, Barts and The London, School of Medicine & Dentistry, QMUL, London, United Kingdom; 2Division of Infection & Immunity, UCL, London, United Kingdom
INTRODUCTION: Treatment strategies in Chronic Hepatitis B (CHB) are now focused on achieving HBsAg loss; therefore greater consideration is being given to combined/sequential therapeutic approaches comprising Pegylated-Interferon (PEG-IFN-α) and nucleot(s)ide analogue (NUC) therapy, to achieve this goal. We previously demonstrated boosting of NK cell responses in eAg- patients treated with PEG-IFN-α (Micco et al, J. Hepatol, 2013), and postulated that this effect could be maintained with sequential NUC therapy, representing a superior strategy to NUC monotherapy. Differential NK cell responses in patients receiving a sequential NUC were compared to patients on NUC monotherapy to determine if there was a treatment advantage with PEG-IFN-α exposure.
PATIENTS & METHODS: PBMC from 18 eAg+ patients during PEG-IFN-α therapy were utlised. 10/18 patients considered PEG-IFN-α non-responders after 48 weeks therapy progressed to sequential NUC therapy and were followed until virally suppressed. NUC monother-apy patients, without prior PEG-IFN-α exposure, were analysed for comparison. Phenotypic and functional analysis of NK cell subsets was performed by multicolour flow-cytometry.
RESULTS: PEG-IFN-α expanded CD56bright NK cells by 3-fold (p=0.0001); this was maintained on sequential therapy but not seen with NUCs alone (p=0.03). NK cell expression of C-Type lectin and natural cytotoxicity receptors was analysed. All receptors, except NKG2D, were expressed at significantly higher levels on sequential NUCs vs. NUC monotherapy (p=<0.05), with marked augmentation in the expression of NKp30 and NKp46 on CD56bright NK cells (p=0.0001 & 0.002 respectively). The proportion of CD56bright NK cells expressing TRAIL was 3-fold higher on sequential NUC therapy compared with NUC monotherapy (p=0.007). These NK cells during sequential therapy demonstrated ability to degranulate and produce IFN-γ; functional restorations not achieved on NUCs alone (p=0.0001 & 0.002 respectively). These changes were more dramatic in patients demonstrating eAg seroconversion +/− sAg decline on sequential NUCs
CONSLUSIONS: The potent expansion of activated CD56bright NK cells induced by PEG-IFN-α is sustained on sequential NUC therapy, with high expression of NKp30, NKp46 and TRAIL when compared to NUCs alone. Restoration of NK cell cytotoxic/effector functions on sequential therapy is seen compared to NUC monother-apy. PEG-IFN-α non-responders exhibit innate boosting which is maintained with functional innate restoration on sequential NUC therapy. Further work is being undertaken to determine if this priming effect is present with shorter courses of PEG-IFN-α.
1708
Functional innate immune responses are restored with sequential NUC therapy following Pegylated Interferon-Alpha exposure and not with NUC monotherapy in Chronic Hepatitis B
Upkar S. Gill1, Dimitra Peppa2, Harsimran D. Singh2, Lorenzo Micco2, Graham R. Foster1, Mala K. Maini2, Patrick T. Kennedy1;
1Hepatology Unit, Centre for Digestive Diseases, Blizard Institute, Barts and The London, School of Medicine & Dentistry, QMUL, London, United Kingdom; 2Division of Infection & Immunity, UCL, London, United Kingdom
INTRODUCTION: Treatment strategies in Chronic Hepatitis B (CHB) are now focused on achieving HBsAg loss; therefore greater consideration is being given to combined/sequential therapeutic approaches comprising Pegylated-Interferon (PEG-IFN-α) and nucleot(s)ide analogue (NUC) therapy, to achieve this goal. We previously demonstrated boosting of NK cell responses in eAg- patients treated with PEG-IFN-α (Micco et al, J. Hepatol, 2013), and postulated that this effect could be maintained with sequential NUC therapy, representing a superior strategy to NUC monotherapy. Differential NK cell responses in patients receiving a sequential NUC were compared to patients on NUC monotherapy to determine if there was a treatment advantage with PEG-IFN-α exposure.
PATIENTS & METHODS: PBMC from 18 eAg+ patients during PEG-IFN-α therapy were utlised. 10/18 patients considered PEG-IFN-α non-responders after 48 weeks therapy progressed to sequential NUC therapy and were followed until virally suppressed. NUC monother-apy patients, without prior PEG-IFN-α exposure, were analysed for comparison. Phenotypic and functional analysis of NK cell subsets was performed by multicolour flow-cytometry.
RESULTS: PEG-IFN-α expanded CD56bright NK cells by 3-fold (p=0.0001); this was maintained on sequential therapy but not seen with NUCs alone (p=0.03). NK cell expression of C-Type lectin and natural cytotoxicity receptors was analysed. All receptors, except NKG2D, were expressed at significantly higher levels on sequential NUCs vs. NUC monotherapy (p=<0.05), with marked augmentation in the expression of NKp30 and NKp46 on CD56bright NK cells (p=0.0001 & 0.002 respectively). The proportion of CD56bright NK cells expressing TRAIL was 3-fold higher on sequential NUC therapy compared with NUC monotherapy (p=0.007). These NK cells during sequential therapy demonstrated ability to degranulate and produce IFN-γ; functional restorations not achieved on NUCs alone (p=0.0001 & 0.002 respectively). These changes were more dramatic in patients demonstrating eAg seroconversion +/− sAg decline on sequential NUCs
CONSLUSIONS: The potent expansion of activated CD56bright NK cells induced by PEG-IFN-α is sustained on sequential NUC therapy, with high expression of NKp30, NKp46 and TRAIL when compared to NUCs alone. Restoration of NK cell cytotoxic/effector functions on sequential therapy is seen compared to NUC monother-apy. PEG-IFN-α non-responders exhibit innate boosting which is maintained with functional innate restoration on sequential NUC therapy. Further work is being undertaken to determine if this priming effect is present with shorter courses of PEG-IFN-α.
" However, this still requires a very long period of treatment as it relies on liver cells turnout to achieve significant reduction of the cccDNA pool, This reduction of cccDNA pool was suggested by a previous abstract that you indicated may have measurement errors. "
The reduction of cccDNA in that abstract was without entry inhibition, you cannot draw conclusions re the speed of infected cell removal as long as they are constantly reinfected. It is a balance, a filling of the bathtub while the drain is open. Stop the filling then you will see the true speed of draining. Thus we do not know how long clearance upon complete blocking of reinfection might take. We do not know the turnover in the infected liver.
So there is complete block of de novo virions or complete block of entry, both of which should lead to clearance depending on the removal speed of infected cells, that we do not really know, but that is also likely quite different in different patients. Both methods are not available now, extremely bad luck prevented myrcludex from becoming that marvelous treatment.
The reduction of cccDNA in that abstract was without entry inhibition, you cannot draw conclusions re the speed of infected cell removal as long as they are constantly reinfected. It is a balance, a filling of the bathtub while the drain is open. Stop the filling then you will see the true speed of draining. Thus we do not know how long clearance upon complete blocking of reinfection might take. We do not know the turnover in the infected liver.
So there is complete block of de novo virions or complete block of entry, both of which should lead to clearance depending on the removal speed of infected cells, that we do not really know, but that is also likely quite different in different patients. Both methods are not available now, extremely bad luck prevented myrcludex from becoming that marvelous treatment.
I would agree a total inhibition of new virions production in combination with drugs such as capsid inhibitors would help tremendously. However, this still requires a very long period of treatment as it relies on liver cells turnout to achieve significant reduction of the cccDNA pool, This reduction of cccDNA pool was suggested by a previous abstract that you indicated may have measurement errors.
As for Myrcludex, it does seem, according to you, that the total blockage required would have too serious side effects on the bile acid re-absorption mechanism to be viable.
Finally, I agree infusion is no big deal to some patients if a finite course can guarantee a cure, but a SVR may not be attractive enough. I am scratching my head to think of another good reason why the progress of REP9AC seems to be so slow.
As for Myrcludex, it does seem, according to you, that the total blockage required would have too serious side effects on the bile acid re-absorption mechanism to be viable.
Finally, I agree infusion is no big deal to some patients if a finite course can guarantee a cure, but a SVR may not be attractive enough. I am scratching my head to think of another good reason why the progress of REP9AC seems to be so slow.
Entry inhibition by blocking NTCP had to be extremely complete to work, as has slso been shown at the recent meeting of the molecular biology of HBV in Los Angeles . Despite 2mg per kg in a humanized mouse trial there was some breakthrough of HDV, quite sobering.
In humans myrcludex dose would need to be probably 50 to 100mg/Day to afford blockage that would lead to infected pool shrinkage.
Since ntcp has to transport literally huge amounts of bile acids from the portal circulation back into the liver, it cannot be blocked to such completion without severe health consequences. Bile acid trapping with cholestyramine is NOT sufficient. The only measure that would work is the surgical installation of a bile duct fistula to the outside, like the poor bears in China have it in the farms for the production of ursodiol from bear bile. It would have to be worn for about a year...then you can block the ntcp to completion.Not a likely treatment the FDA would easily approve, I am afraid...
In humans myrcludex dose would need to be probably 50 to 100mg/Day to afford blockage that would lead to infected pool shrinkage.
Since ntcp has to transport literally huge amounts of bile acids from the portal circulation back into the liver, it cannot be blocked to such completion without severe health consequences. Bile acid trapping with cholestyramine is NOT sufficient. The only measure that would work is the surgical installation of a bile duct fistula to the outside, like the poor bears in China have it in the farms for the production of ursodiol from bear bile. It would have to be worn for about a year...then you can block the ntcp to completion.Not a likely treatment the FDA would easily approve, I am afraid...
Just lost a long explanatory post..so now in brief:
A strong further reduction in HBV replication beyond the power of nucs would finally limit the spread of HBV that occurs even on Tdf. A capsid Formation inhibitor of high effectiveness could work in Synergy with nucs. No more de Novo virions would mean no more spreading.
High efficiency blocking of hbsag Production or release will remove a strong HBV defense system to secure its continued existence in a chronically infected human.
We have the NAPs, they work miraculously well in that regard. A weekly infusion is nothing, it is just a little hard to sell in an overformalized medical care system. But remicade patients get infusions all the time.
A strong further reduction in HBV replication beyond the power of nucs would finally limit the spread of HBV that occurs even on Tdf. A capsid Formation inhibitor of high effectiveness could work in Synergy with nucs. No more de Novo virions would mean no more spreading.
High efficiency blocking of hbsag Production or release will remove a strong HBV defense system to secure its continued existence in a chronically infected human.
We have the NAPs, they work miraculously well in that regard. A weekly infusion is nothing, it is just a little hard to sell in an overformalized medical care system. But remicade patients get infusions all the time.
Those are nice and interesting lab results concerning important immunological parameters in ifn and tenofovir sequential patients, quite encouraging.
But those shifts in immune cells activity need to be translated into clinical success. It is currently not clear what the optimal nuc pretreatment time is to let the immunity recuperate to a more functional state with a higher chance of subsequent activation of effective elimination mechanisms. As we have seen, the shorter term combo and sequential trials do not have high hbsag loss rates.
But those shifts in immune cells activity need to be translated into clinical success. It is currently not clear what the optimal nuc pretreatment time is to let the immunity recuperate to a more functional state with a higher chance of subsequent activation of effective elimination mechanisms. As we have seen, the shorter term combo and sequential trials do not have high hbsag loss rates.
1740
Nucleotide analogue improves interferon responsiveness in HBV-infected human hepatocytes
Masataka Tsuge1,3, Nobuhiko Hiraga1,2, Eisuke Murakami1,2, Michio Imamura1,2, Hiromi Abe1,2, Daiki Miki1,2, Hidenori Ochi1,2, C. Nelson Hayes1,2, Kazuaki Chayama1,2;
1Department of Gastroenterology and Metabolism, Hiroshima university, Hiroshima, Japan; 2Liver Research Project Center, Hiroshima university, Hiroshima, Japan; 3Natural Science Center for Basic Research and Development, Hiroshima university, Hiroshima, Japan
Background: It has been reported that interferon treatment could reduce HBs antigen (HBsAg) production in patients with chronic hepatitis B virus (HBV) infection. However, only limited HBsAg reduction is observed in patients treated with interferon therapy. One cause of this limitation may be that interferon responsiveness in human hepatocytes is suppressed by HBV infection, and, therefore, interferon stimulated genes (ISGs) are not induced sufficiently to promote anti-viral effects. In the present study, we analyzed whether the suppression of HBV replication using nucleotide analogues (NAs) could improve interferon responsiveness in HBV infected human hepatocytes. Methods: Thirty-seven chronic hepatitis B patients were enrolled. Twenty patients underwent sequential interferon therapy, which included 6 months of conventional interferon therapy, running from one month prior to discontinuation until 5 months after discontinuation of NA therapy. The remaining 17 patients underwent interferon mono-therapy. Serum HBsAg titers were measured every year for 5 years after interferon therapy. To confirm the clinical results, we performed an in vitro study using T23 cells, which were generated from HepG2 cells stably transfected with an HBV expression plasmid. T23 cells were treated with or without the NA entecavir for 5 days. The cells were then treated with IFN for 6 hours and harvested. To evaluate the interferon responsiveness, ISG mRNA levels were quantified by real time PCR. Results: In the clinical study, more than 1 Log IU/ml reduction of HBsAg titer was achieved in 11 of 37 patients (interferon mono-therapy: 2, Sequential therapy: 9). By univariate analysis, the following factors, gender, serum HBsAg level, the existence of HBeAg, and prior NA therapy, were associated with HBsAg reduction (P=0.007, P=0.027, P=0.031, P=0.037, respectively). From the clinical results, it was predicted that interferon responsiveness might be improved by prior NA therapy. To verify these results, in vitro experiments were performed. In the absence of HBV, the ISGs MxA and OAS1 were significantly induced by interferon treatment (19.2-fold, 9.7-fold, respectively). However, in T23 cells, inductions of these ISGs was suppressed (P=0.0495, P=0.0495, respectively). After entecavir treatment, interferon responsiveness was restored and ISG induction increased (P=0.0495, P=0.0339, respectively). Conclusions: Prior NA therapy could improve interferon responsiveness in HBV infected human hepatocytes. To improve the anti-viral effects in chronic hepatitis B patients, it might be necessary to revise the way of using NAs and interferons.
Nucleotide analogue improves interferon responsiveness in HBV-infected human hepatocytes
Masataka Tsuge1,3, Nobuhiko Hiraga1,2, Eisuke Murakami1,2, Michio Imamura1,2, Hiromi Abe1,2, Daiki Miki1,2, Hidenori Ochi1,2, C. Nelson Hayes1,2, Kazuaki Chayama1,2;
1Department of Gastroenterology and Metabolism, Hiroshima university, Hiroshima, Japan; 2Liver Research Project Center, Hiroshima university, Hiroshima, Japan; 3Natural Science Center for Basic Research and Development, Hiroshima university, Hiroshima, Japan
Background: It has been reported that interferon treatment could reduce HBs antigen (HBsAg) production in patients with chronic hepatitis B virus (HBV) infection. However, only limited HBsAg reduction is observed in patients treated with interferon therapy. One cause of this limitation may be that interferon responsiveness in human hepatocytes is suppressed by HBV infection, and, therefore, interferon stimulated genes (ISGs) are not induced sufficiently to promote anti-viral effects. In the present study, we analyzed whether the suppression of HBV replication using nucleotide analogues (NAs) could improve interferon responsiveness in HBV infected human hepatocytes. Methods: Thirty-seven chronic hepatitis B patients were enrolled. Twenty patients underwent sequential interferon therapy, which included 6 months of conventional interferon therapy, running from one month prior to discontinuation until 5 months after discontinuation of NA therapy. The remaining 17 patients underwent interferon mono-therapy. Serum HBsAg titers were measured every year for 5 years after interferon therapy. To confirm the clinical results, we performed an in vitro study using T23 cells, which were generated from HepG2 cells stably transfected with an HBV expression plasmid. T23 cells were treated with or without the NA entecavir for 5 days. The cells were then treated with IFN for 6 hours and harvested. To evaluate the interferon responsiveness, ISG mRNA levels were quantified by real time PCR. Results: In the clinical study, more than 1 Log IU/ml reduction of HBsAg titer was achieved in 11 of 37 patients (interferon mono-therapy: 2, Sequential therapy: 9). By univariate analysis, the following factors, gender, serum HBsAg level, the existence of HBeAg, and prior NA therapy, were associated with HBsAg reduction (P=0.007, P=0.027, P=0.031, P=0.037, respectively). From the clinical results, it was predicted that interferon responsiveness might be improved by prior NA therapy. To verify these results, in vitro experiments were performed. In the absence of HBV, the ISGs MxA and OAS1 were significantly induced by interferon treatment (19.2-fold, 9.7-fold, respectively). However, in T23 cells, inductions of these ISGs was suppressed (P=0.0495, P=0.0495, respectively). After entecavir treatment, interferon responsiveness was restored and ISG induction increased (P=0.0495, P=0.0339, respectively). Conclusions: Prior NA therapy could improve interferon responsiveness in HBV infected human hepatocytes. To improve the anti-viral effects in chronic hepatitis B patients, it might be necessary to revise the way of using NAs and interferons.
1735
Phenotypic Characteristics of PD-1, CTLA-4 and FoxP3 Expression during Tenofovir therapy in Chronic Hepatitis B
Hyosun Cho1, Chang Wook Kim2, Yu seung Kim2, Hee Yeon Kim2, Jong Young Choi2, Seung Kew Yoon2, Chang Don Lee3;
1Pharmacy, Duksung Women's University, Seoul, Republic of Korea; 2Internal Medicine, The Catholic University of Korea, Uijeonbu-shi, Republic of Korea; 3Internal Medicine, International St. Mary's Hospital, Incheon, Republic of Korea
Background: Inhibitory molecules such as programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) are associated with antiviral effector T-cell dysfunction, which influences on T-cell exhaustion and persistent viral infection. These PD-1 and CTLA-4 are up-regulated in chronic viral infection such as chronic hepatitis C, chronic hepatitis B and human immunodeficiency virus infection but there is few report about the role of PD-1 and CTLA-4 in patients with chronic hepatitis B during antiviral therapy with tenofovir. We investigated the expression of PD-1 and CTLA-4 during tenofovir treatment in patients with chronic hepatitis B. Methods: Nine patients with chronic hepatitis B under tenofovir treatment were enrolled for detection of intrinsic inhibitory molecules of T cell signals (PD-1, CTLA-4) and extrinsic inhibitory molecule, FoxP3. Peripheral blood mononuclear cells (PBMC) were isolated from these subjects before tenofovir treatment (T0) and 1 month (T1), 3 month (T3), 6 month (T6) during tenofovir treatment. The expressions of PD-1, CTLA-4 and FoxP3 on T cells were monitored by flow cytometry. Results: T cells from patients with chronic hepatitis B under tenofovir treatment showed decreased expression of PD-1, CTLA-4, and FoxP3 at T6 compared to T0 (%PD-1/ CD8, 5.0 ± 2.2 vs. 4.0 ± 1.2; %CTLA-4/CD8, 1.7 ± 0.9 vs. 1.2 ± 0.6; %FoxP3/CD4, 7 ± 2.5 vs. 6.1 ± 2.6 showed as mean ± SD). During the initial phase of tenofovir treatment, FoxP3 and PD-1 fluctuate at T1 and T3 but, CTLA-4 decreased steadily even at T1 and T3. Conclusions: In chronic hepatitis B, PD-1 and CTLA-4 as inhibitory T cell molecules and FoxP3 as regulatory T cell marker are down-regulated during initial 6 month tenofovir therapy, which could restore HBV-specific T cell function during tenofovir antiviral therapy. Long-term effects of tenofovir on host immune system are needed to be elucidate.
Phenotypic Characteristics of PD-1, CTLA-4 and FoxP3 Expression during Tenofovir therapy in Chronic Hepatitis B
Hyosun Cho1, Chang Wook Kim2, Yu seung Kim2, Hee Yeon Kim2, Jong Young Choi2, Seung Kew Yoon2, Chang Don Lee3;
1Pharmacy, Duksung Women's University, Seoul, Republic of Korea; 2Internal Medicine, The Catholic University of Korea, Uijeonbu-shi, Republic of Korea; 3Internal Medicine, International St. Mary's Hospital, Incheon, Republic of Korea
Background: Inhibitory molecules such as programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) are associated with antiviral effector T-cell dysfunction, which influences on T-cell exhaustion and persistent viral infection. These PD-1 and CTLA-4 are up-regulated in chronic viral infection such as chronic hepatitis C, chronic hepatitis B and human immunodeficiency virus infection but there is few report about the role of PD-1 and CTLA-4 in patients with chronic hepatitis B during antiviral therapy with tenofovir. We investigated the expression of PD-1 and CTLA-4 during tenofovir treatment in patients with chronic hepatitis B. Methods: Nine patients with chronic hepatitis B under tenofovir treatment were enrolled for detection of intrinsic inhibitory molecules of T cell signals (PD-1, CTLA-4) and extrinsic inhibitory molecule, FoxP3. Peripheral blood mononuclear cells (PBMC) were isolated from these subjects before tenofovir treatment (T0) and 1 month (T1), 3 month (T3), 6 month (T6) during tenofovir treatment. The expressions of PD-1, CTLA-4 and FoxP3 on T cells were monitored by flow cytometry. Results: T cells from patients with chronic hepatitis B under tenofovir treatment showed decreased expression of PD-1, CTLA-4, and FoxP3 at T6 compared to T0 (%PD-1/ CD8, 5.0 ± 2.2 vs. 4.0 ± 1.2; %CTLA-4/CD8, 1.7 ± 0.9 vs. 1.2 ± 0.6; %FoxP3/CD4, 7 ± 2.5 vs. 6.1 ± 2.6 showed as mean ± SD). During the initial phase of tenofovir treatment, FoxP3 and PD-1 fluctuate at T1 and T3 but, CTLA-4 decreased steadily even at T1 and T3. Conclusions: In chronic hepatitis B, PD-1 and CTLA-4 as inhibitory T cell molecules and FoxP3 as regulatory T cell marker are down-regulated during initial 6 month tenofovir therapy, which could restore HBV-specific T cell function during tenofovir antiviral therapy. Long-term effects of tenofovir on host immune system are needed to be elucidate.
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