Developments in Cell-Penetrating Peptides as Antiviral Agents and as Vehicles for Delivery of Peptide Nucleic Acid Targeting Hepadnaviral Replication Pathway.

Alternative therapeutic approaches against chronic hepatitis B virus (HBV) infection need to be urgently developed because current therapies are only virostatic. In this context, cell penetration peptides (CPPs) and their Peptide Nucleic Acids (PNAs) cargoes appear as a promising novel class of biologically active compounds. In this review we summarize different in vitro and in vivo studies, exploring the potential of CPPs as vehicles for intracellular delivery of PNAs targeting hepadnaviral replication. Thus, studies conducted in the duck HBV (DHBV) infection model showed that conjugation of (D-Arg)8 CPP to PNA targeting viral epsilon (ε) were able to efficiently inhibit viral replication in vivo following intravenous administration to ducklings. Unexpectedly, some CPPs, (D-Arg)8 and Decanoyl-(D-Arg)8, alone displayed potent antiviral effect, altering late stages of DHBV and HBV morphogenesis. Such antiviral effects of CPPs may affect the sequence-specificity of CPP-PNA conjugates. By contrast, PNA conjugated to (D-Lys)4 inhibited hepadnaviral replication without compromising sequence specificity. Interestingly, Lactose-modified CPP mediated the delivery of anti-HBV PNA to human hepatoma cells HepaRG, thus improving its antiviral activity. In light of these promising data, we believe that future studies will open new perspectives for translation of CPPs and CPP-PNA based technology to therapy of chronic hepatitis B.

Nucleic acid polymer REP 2139 and nucleos(T)ide analogues act synergistically against chronic hepadnaviral infection in vivo in Pekin ducks.

Nucleic acid polymer (NAP) REP 2139 treatment was shown to block the release of viral surface antigen in duck HBV (DHBV)-infected ducks and in patients with chronic HBV or HBV/hepatitis D virus infection. In this preclinical study, a combination therapy consisting of REP 2139 with tenofovir disoproxil fumarate (TDF) and entecavir (ETV) was evaluated in vivo in the chronic DHBV infection model. DHBV-infected duck groups were treated as follows: normal saline (control); REP 2139 TDF; REP 2139 + TDF; and REP 2139 + TDF + ETV. After 4 weeks of treatment, all animals were followed for 8 weeks. Serum DHBsAg and anti-DHBsAg antibodies were monitored by enzyme-linked immunosorbent assay and viremia by qPCR. Total viral DNA and covalently closed circular DNA (cccDNA) were quantified in autopsy liver samples by qPCR. Intrahepatic DHBsAg was assessed at the end of follow-up by immunohistochemistry. On-treatment reduction of serum DHBsAg and viremia was more rapid when REP 2139 was combined with TDF or TDF and ETV, and, in contrast to TDF monotherapy, no viral rebound was observed after treatment cessation. Importantly, combination therapy resulted in a significant decrease in intrahepatic viral DNA (>3 log) and cccDNA (>2 log), which were tightly correlated with the clearance of DHBsAg in the liver. CONCLUSION: Synergistic antiviral effects were observed when REP 2139 was combined with TDF or TDF + ETV leading to control of infection in blood and liver, associated with intrahepatic viral surface antigen elimination that persisted after treatment withdrawal. Our findings suggest the potential of developing such combination therapy for treatment of chronically infected patients in the absence of pegylated interferon. (Hepatology 2018;67:2127-2140).

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication.

Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA-targeting hepadnaviral encapsidation signal (ε). This anti-ε PNA exhibited sequence-specific inhibition of DHBV RT in a cell-free system. Investigation of the best in vivo route of delivery of PNA conjugated to (D-Arg)8 (P1) showed that intraperitoneal injection to ducklings was ineffective, whereas intravenously (i.v.) injected fluorescein-P1-PNA reached the hepatocytes. Treatment of virus carriers with i.v.-administered P1-PNA resulted in a decrease in viral DNA compared to untreated controls. Surprisingly, a similar inhibition of viral replication was observed in vivo as well as in vitro in primary hepatocyte cultures for a control 2 nt mismatched PNA conjugated to P1. By contrast, the same PNA coupled to (D-Lys)4 (P2) inhibited DHBV replication in a sequence-specific manner. Interestingly, only P1, but not P2, displayed anti-DHBV activity in the absence of PNA cargo. Hence, we provide new evidence that CPP-PNA conjugates inhibit DHBV replication following low-dose administration. Importantly, our results demonstrate the key role of CPPs used as vehicles in antiviral specificity of CPP-PNA conjugates.

Nucleic Acid Polymers with Accelerated Plasma and Tissue Clearance for Chronic Hepatitis B Therapy.

REP 2139 is a nucleic acid polymer (NAP) currently under clinical development for chronic hepatitis B (HBV) therapy. This preclinical study investigated different REP 2139 analogs that would display reduced accumulation in the serum and tissues, while retaining an antiviral effect against HBV infection. REP 2139 analogs were evaluated in human plasma, CD-1 mice, cynomolgus monkeys, and Pekin ducks. Discrete ribose transformation to 2'OH in selected riboadenosines resulted in a slow degradation in acidified human plasma that plateaued after 48 hr. REP 2165, a REP 2139 analog containing three unmodified riboadenosines equally spaced throughout the polymer, showed similar plasma clearance and tissue distribution as REP 2139 in mice and cynomolgus monkeys after a single dose. Interestingly, after repeated administration, accumulation of REP 2165 in plasma and organs was reduced, indicating a dramatically faster rate of clearance from organs after therapy was ended in both species. Both REP 2139 and REP 2165 were well tolerated at clinically relevant doses, with no alterations in liver, kidney, or hematological function. In chronic duck HBV (DHBV) infection, REP 2165 displayed significantly reduced liver accumulation after repeated dosing but retained antiviral activity similar to REP 2139. These results indicate the therapeutic potential of REP 2165 against chronic HBV infection in patients is similar to REP 2139, but with significantly reduced drug accumulation and improved tissue clearance.

Present and future DNA vaccines for chronic hepatitis B treatment.

INTRODUCTION: With at least 240 million hepatitis B virus (HBV) carriers worldwide, being at a high risk of cirrhosis and hepatocellular carcinoma (HCC), chronic hepatitis B remains a major public health issue. Because current antiviral treatments are only virostatic, there is an urgent need for the development of innovative anti-HBV strategies leading to the functional cure. In this context, DNA-based vaccines appear as a promising approach. Area covered: In this review, the authors summarize the pertinent features of DNA vaccines for chronic hepatitis B therapy. They review several technologies that improve DNA vaccines efficacy evaluated in animal models of hepadnaviral infection. They also discuss the clinical trials of therapeutic DNA vaccination initiated in HBV-carrier patients. Expert opinion: Preclinical studies in HBV transgenic mice, DHBV-carrier ducks and WHV-infected woodchucks, have clearly demonstrated a benefit of DNA vaccine-based combination therapies for chronic hepatitis B treatment. However, the results of clinical trials conducted in HBV patients were rather disappointing and frustrating, as DNA-vaccines have not shown the same efficacy in patients as in animal models. We are convinced that the design of innovative clinical trials based on strategies able to increase DNA vaccine immunogenicity will allow to advance in this challenging field.

Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B.

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B.

Advances and challenges in the development of therapeutic DNA vaccines against hepatitis B virus infection.

Despite the existence of an effective prophylactic vaccine, chronic hepatitis B virus (HBV) infection remains a major public health problem. Because very weak and functionally impaired virus-specific immune responses play a key role in the persistence of HBV infection, the stimulation of these responses appears to be of particular importance for virus clearance. In this regard DNA-based vaccination has emerged as novel, promising therapeutic approach for chronic hepatitis B. This review provides an update of preclinical studies in animal models (mouse, chimpanzee, duck, woodchuck), which evaluated the ability of DNA vaccines targeting hepadnaviral proteins to induce potent and sustained immune responses in naïve animals and to enhance virus clearance and break immune tolerance in chronic virus-carriers. Different strategies have been developed and evaluated in these models to optimize DNA vaccine including genetic adjuvants, combination with antiviral drugs, prime-boost regimens and plasmid delivery. The delivery of DNA by in vivo electroporation appears to be of particular interest for increase of vaccine potency in both small and large animal models. Based on the promising results generated in preclinical studies, first clinical trials of DNA vaccines have been initiated, although effective therapy of chronic hepatitis B awaits further improvements in vaccine efficacy.

Dual inhibition of HCV and HIV by ring-expanded nucleosides containing the 5:7-fused imidazo[4,5-e][1,3]diazepine ring system. In vitro results and implications.

Examples of ring-expanded nucleosides (RENs), represented by general structures 1 and 2, exhibited dual anti-HCV and anti-HIV activities in both cell culture systems and the respective target enzyme assays, including HCV NTPase/helicase and human RNA helicase DDX3. Since HCV is a leading co-infection in late stage HIV AIDS patients, often leading to liver cirrhosis and death, the observed dual inhibition of HCV and HIV by the target nucleoside analogues has potentially beneficial implications in treating HIV patients infected with HCV.

Discovery of naturally occurring transmissible chronic hepatitis B virus infection among Macaca fascicularis from Mauritius Island.

UNLABELLED: Despite a high prevalence of hepatitis B virus (HBV) infection in endangered apes, no HBV infection has been reported in small, old-world monkeys. In search for a small, nonhuman primate model, we investigated the prevalence of HBV infection in 260 macaque (Cercopithecidae) sera of various geographical origins (i.e., Morocco, Mauritius Island, and Asia). HBV-positive markers were detected in cynomolgus macaques (Macaca fascicularis) from Mauritius Island only, and, remarkably, HBV DNA was positive in 25.8% (31 of 120) and 42% (21 of 50) of serum and liver samples, respectively. Strong liver expression of hepatitis B surface antigen and hepatitis B core antigen was detected in approximately 20%-30% of hepatocytes. Furthermore, chronic infection with persisting HBV DNA was documented in all 6 infected macaques during an 8-month follow-up period. Whole HBV genome-sequencing data revealed that it was genotype D subtype ayw3 carrying substitution in position 67 of preS1. To confirm infectivity of this isolate, 3 Macaca sylvanus were inoculated with a pool of M. fascicularis serum and developed an acute HBV infection with 100% sequence homology, compared with HBV inoculum. We demonstrated the presence of a chronic HBV infection in M. fascicularis from Mauritius Island. This closely human-related HBV might have been transmitted from humans, because the initial breeding colony originated from very few ancestors 300 years ago when it was implemented by Portuguese who imported a handful of macaques from Java to Mauritius Island. CONCLUSION: This report on natural, persisting HBV infection among cynomolgus macaques provides the first evidence for the existence of a novel, small simian model of chronic HBV infection, immunologically close to humans, that should be most valuable for the study of immunotherapeutic approaches against chronic hepatitis B.

Potent inhibition of late stages of hepadnavirus replication by a modified cell penetrating peptide.

Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication of Duck Hepatitis B Virus (DHBV), a reference model for human HBV. Amongst twelve CatLip peptides we identified Deca-(Arg)8 having a particularly potent antiviral activity, leading to a drastic inhibition of viral particle secretion without detectable toxicity. Inhibition of virion secretion was correlated with a dose-dependent increase in intracellular viral DNA. Deca-(Arg)8 peptide did neither interfere with DHBV entry, nor with formation of mature nucleocapsids nor with their travelling to the nucleus. Instead, Deca-(Arg)8 caused envelope protein accumulation in large clusters as revealed by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)8-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis via interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside the mode of inhibition. Deca-(Arg)8 may be a useful tool for elucidating the hepadnaviral secretory pathway, which is not yet fully understood. Moreover we provide the first evidence that a modified CPP displays a novel antiviral mechanism targeting another step of viral life cycle compared to what has been so far described for other enveloped viruses.

In vivo electroporation improves therapeutic potency of a DNA vaccine targeting hepadnaviral proteins.

This preclinical study investigated the therapeutic efficacy of electroporation (EP)-based delivery of plasmid DNA (pDNA) encoding viral proteins (envelope, core) and IFN-γ in the duck model of chronic hepatitis B virus (DHBV) infection. Importantly, only DNA EP-therapy resulted in a significant decrease in mean viremia titers and in intrahepatic covalently closed circular DNA (cccDNA) levels in chronic DHBV-carrier animals, compared with standard needle pDNA injection (SI). In addition, DNA EP-therapy stimulated in all virus-carriers a humoral response to DHBV preS protein, recognizing a broader range of major antigenic regions, including neutralizing epitopes, compared with SI. DNA EP-therapy led also to significant higher intrahepatic IFN-γ RNA levels in DHBV-carriers compared to other groups, in the absence of adverse effects. We provide the first evidence on DNA EP-therapy benefit in terms of hepadnaviral infection clearance and break of immune tolerance in virus-carriers, supporting its clinical application for chronic hepatitis B.

Enhanced magnitude and breadth of neutralizing humoral response to a DNA vaccine targeting the DHBV envelope protein delivered by in vivo electroporation.

We explored in the duck hepatitis B virus (DHBV) model the impact of electroporation (EP)-mediated DNA vaccine delivery on the neutralizing humoral response to viral preS/S large envelope protein. EP enhanced the kinetics and magnitude of anti-preS response compared to the standard needle DNA injection (SI). Importantly, EP dramatically enhanced the neutralizing potency of the humoral response, since antibodies induced by low DNA dose (10 µg) were able to highly neutralize DHBV and to recognize ten antigenic regions, including four neutralization epitopes. Whereas, SI-induced antibodies by the same low DNA dose were not neutralizing and the epitope pattern was extremely narrow, since it was limited to only one epitope. Thus, EP-based delivery was able to improve the dose efficiency of DNA vaccine and to maintain a highly neutralizing, multi-specific B-cell response, suggesting that it may be an effective approach for chronic hepatitis B therapy at clinically feasible DNA dose.

Enhancement of neutralizing humoral response of DNA vaccine against duck hepatitis B virus envelope protein by co-delivery of cytokine genes.

We explored in the duck hepatitis B virus (DHBV) model the impact of duck interferon gamma (Du-IFNgamma) or interleukin 2 (Du-IL2) co-delivery on humoral neutralizing response induced by DNA-based vaccine encoding DHBV preS/S large envelope protein. Co-delivery of either Du-IL2 or Du-IFNgamma encoding plasmids considerably increased the magnitude of anti-preS humoral response. Moreover, co-administration of cytokine genes led to a significant (p<0.001) enhancement of neutralizing anti-DHBV antibody response, which was more pronounced for Du-IFNgamma. Our data suggest that co-delivery of cytokine and envelope protein encoding plasmids will be a valuable approach for the development of a potent therapeutic DNA vaccine against chronic hepatitis B.

DNA vaccination in combination or not with lamivudine treatment breaks humoral immune tolerance and enhances cccDNA clearance in the duck model of chronic hepatitis B virus infection.

This study used a duck hepatitis B virus (DHBV) model to evaluate whether a novel DNA vaccination protocol alone or associated with antiviral (lamivudine) treatment was able to clear the intrahepatic covalently closed, circular viral DNA (cccDNA) pool responsible for persistence of infection. DHBV carriers received DNA vaccine (on weeks 6, 10, 13, 14, 28 and 35) targeting the large envelope and/or core proteins alone or combined with lamivudine treatment (on weeks 1-8) or lamivudine monotherapy. After 10 months of follow-up, a dramatic decrease in viraemia and liver DHBV cccDNA (below 0.08 cccDNA copies per cell) was observed in 9/30 ducks (30 %) receiving DNA mono- or combination therapy, compared with 0/12 (0 %) from lamivudine monotherapy or the control groups, suggesting a significant antiviral effect of DNA immunization. However, association with the drug did not significantly improve DHBV DNA vaccine efficacy (33 % cccDNA clearance for the combination vs 27 % for DNA monotherapy), probably due to the low antiviral potency of lamivudine in the duck model. Seroconversion to anti-preS was observed in 6/9 (67 %) ducks showing cccDNA clearance, compared with 1/28 (3.6 %) without clearance, suggesting a significant correlation (P<0.001) between humoral response restoration and cccDNA elimination. Importantly, an early (weeks 10-12) drop in viraemia was observed in seroconverted animals, and virus replication did not rebound following the cessation of immunotherapy, indicating a sustained effect. This study provides the first evidence that therapeutic DNA vaccination is able to enhance hepadnaviral cccDNA clearance, which is tightly associated with a break in humoral immune tolerance. These results also highlight the importance of antiviral drug potency and an effective DNA immunization protocol for the design of therapeutic vaccines against chronic hepatitis B.

Bicistronic woodchuck hepatitis virus core and gamma interferon DNA vaccine can protect from hepatitis but does not elicit sterilizing antiviral immunity.

The immunity elicited against nucleocapsid of hepatitis B virus (HBV) and closely related woodchuck hepatitis virus (WHV) has been shown to be important in resolution of hepatitis and protection from infection. Further, activity of gamma interferon (IFN-gamma), which may directly inhibit hepadnavirus replication, promotes antiviral defense and favors T helper cell type 1 (Th1) response, which is seemingly a prerequisite of HBV clearance. In this study, to enhance induction of protective immunity against hepadnavirus, healthy woodchucks were immunized with a bicistronic DNA vaccine carrying WHV core (WHc) and woodchuck IFN-gamma (wIFN-gamma) gene sequences. Three groups, each group containing three animals, were injected once or twice with 0.5 mg, 0.9 mg, or 1.5 mg per dose of this vaccine. In addition, four animals received two injections of 0.6 mg or 1 mg WHc DNA alone. All animals were challenged with WHV. The results showed that four of nine animals injected with the bicistronic vaccine and one of four immunized with WHc DNA became protected from serologically evident infection and hepatitis. This protection was not linked to induction of WHc antigen-specific antibodies or T-cell proliferative response and was not associated with enhanced transcription of Th1 cytokines or 2',5'-oligoadenylate synthetase. Strikingly, all animals protected from hepatitis became reactive for WHV DNA and carried low levels of replicating virus in hepatic and lymphoid tissues after challenge with WHV. This study shows that the bicistronic DNA vaccine encoding both hepadnavirus core antigen and IFN-gamma was more effective in preventing hepatitis than that encoding virus core alone, but neither of them could mount sterile immunity against the virus or prevent establishment of occult infection.

Rise in gamma interferon expression during resolution of duck hepatitis B virus infection.

Gamma interferon (IFN-gamma) expression plays a crucial role in the control of mammalian hepatitis B virus (HBV) infection. However, the role of duck INF-gamma (DuIFN-gamma) in the outcome of duck HBV (DHBV) infection, a reference model for hepadnavirus replication studies, has not yet been investigated. This work explored the dynamics of DuIFN-gamma expression in liver and peripheral blood mononuclear cells (PBMCs) during resolution of DHBV infection in adolescent ducks in relation to serum and liver markers of virus replication, histological changes and humoral response induction. DHBV infection of 3-week-old ducks resulted in transient expression of intrahepatic preS protein (days 3-14) and mild histological changes. Low-level viraemia was detected only during the first 10 days of infection and was accompanied by early anti-preS antibody response induction. Importantly, a strong increase in intrahepatic DuIFN-gamma RNA was detected by real-time RT-PCR at days 6-14, which coincided with a sharp decrease in both viral DNA and preS protein in the liver. Interestingly, liver DuIFN-gamma expression remained augmented to the end of the follow-up period (day 66) and correlated with portal lymphocyte infiltration and persistence of trace quantities of intrahepatic DHBV DNA in animals that had apparently completely resolved the infection. Moreover, in infected ducks, a moderate increase was detected in the levels of DuIFN-gamma in PBMCs (days 12-14), which coincided with the peak in liver DuIFN-gamma RNA levels. These data reveal that increased DuIFN-gamma expression in liver and PBMCs is concomitant with viral clearance, characterizing the resolution of infection, and provide new insights into the host-virus interactions that control DHBV infection.

Genetic vaccination for production of DNA-designed antibodies specific to Hepadnavirus envelope proteins.

We propose a method of avian antibodies production based on DNA immunization of laying ducks with a plasmid encoding specified antigen, followed by egg collection and purification of egg yolk immunoglobulins (IgY). We have validated this approach in the Duck hepatitis B virus (DHBV) model. We report here that following immunization of female ducks with plasmids encoding DHBV envelope proteins, large amounts (at least 50 mg/egg) of specific antibodies can be obtained from eggs of these ducks. Interestingly, the comparison of different plasmid constructs showed the important differences in their efficacy of specific IgY antibodies induction in the sera and eggs of immunized ducks.

Genetic immunization of ducks for production of antibodies specific to Helicobacter pylori UreB in egg yolks.

Following genetic immunization of laying ducks with a plasmid expressing Helicobacter pylori UreB (large subunit of urease), IgY against UreB were obtained from egg yolks. These polyclonal and monospecific IgY antibodies are of higher-titer and specifically recognize recombinant H. pylori urease purified from Escherichia coli. To our knowledge this is the first report describing generation of IgY antibodies directed against antigens of H. pylori by DNA-based immunization.

Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA).

BACKGROUND/AIMS: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT) and to compare their efficacy with phosphorothioate oligodeoxynucleotides (S-ODNs). METHODS: The effect of two partly overlapping PNAs targeting epsilon and of analogous S-ODNs was tested in cell-free transcription and translation system for DHBV RT expression. In addition their antiviral effect was investigated in primary duck hepatocytes (PDH). RESULTS: Both PNAs reproducibly inhibited DHBV RT in a dose-dependent manner with IC(50) of 10nM, whereas up to 600-fold higher concentration of S-ODNs was required for similar inhibition. The PNA targeting the bulge and upper stem of epsilon appeared as more efficient RT inhibitor than the PNA targeting only the bulge. Importantly, the inhibition was highly sequence-specific since double-mismatched PNA had no effect on the RT reaction. Moreover, in PDH the PNA coupled to Arg(7) cationic delivery peptide decreased DHBV replication. CONCLUSIONS: We provide the first evidence that PNAs targeting the bulge and upper stem of epsilon can efficiently and in a sequence-specific manner inhibit DHBV RT.