Summer Academy “Infection and Immunity in times of COVID ...€¦ · the evolutionary relationships among highly diverged sequences. In our analysis for HDV-1, we observed amino

Summer Academy “Infection and Immunity in times of COVID-19” Lecture on Wednesday, 19 August 2020

Prof. Dr. Michael Roggendorf

Pathogenesis of hepatitis D Virus: the most severe form of chronic hepatitis Publications for seminar preparation: Zainab Usman , Stoyan Velkov , Ulrike Protzer , Michael Roggendorf, Dmitrij Frishman, Hadi Karimzadeh HDVdb: A Comprehensive Hepatitis D Virus Database Viruses. 2020 May 14;12(5):538. doi: 10.3390/v12050538. Hadi Karimzadeh, Zainab Usman, Dmitrij Frishman , Michael Roggendorf Genetic Diversity of Hepatitis D Virus genotype-1 in Europe Allows Classification Into Subtypes J Viral Hepat. 2019 Jul;26(7):900-910. doi: 10.1111/jvh.13086. Epub 2019 Mar 15. Hadi Karimzadeh et al. Mutations in Hepatitis D Virus Allow It to Escape Detection by CD8 + T Cells and Evolve at the Population Level Gastroenterology. 2019 May;156(6):1820-1833. doi: 10.1053/j.gastro.2019.02.003. Epub 2019 Feb 12. Hadi Karimzadeh et al. Amino Acid Substitutions Within HLA-B*27-Restricted T Cell Epitopes Prevent Recognition by Hepatitis Delta Virus-Specific CD8 + T Cells J Virol. 2018 Jun 13;92(13):e01891-17. doi: 10.1128/JVI.01891-17. Print 2018 Jul 1. And the following two reviews:

Technical University Munich Institute of Virology [email protected] [email protected]

mailto:[email protected]:[email protected]

125

ABSTRACT

To date, the strains of the Hepatitis D (Delta) Virus (HDV) identified worldwide have been classified into eight major genotypes (HDV-1 to -8). HDV-1 is the most prevalent genotype worldwide, whereas HDV-2 to -8 genotypes are, in some cases exclusively, distributed in specific geographical regions.Although HDV-1 is found in many different parts of the world, detailed genetic analysis can define region-specific distribution of subtypes of HDV-1 with certain evolutionary distances. Here, we discuss subtyping of European genotype 1 isolates of HDV as an example, and ex-plain how reducing redundant sequences can facilitate a detailed classification of HDV into subtypes with unique evolutionary distances and distinct geographical distribution. Based on the lessons learned in the case of other hepatitis viruses such as Hepatitis C Virus (HCV), we suggest that sub-classification of HDV might be helpful for better understanding of subtype-related pathogenesis, clinical symptoms, response to therapy, replication capacity and transmission efficiency.

Key words: Hepatitis D (Delta) Virus, large Hepatitis Delta Antigen, inter-subtype, geno-typing.

Introduction

The Hepatitis D (Delta) Virus (HDV) is a defective RNA virus that requires the HBsAg provided by the Hepatitis B Virus (HBV) for transmission.1 The virion con-tains a circular single-stranded RNA genome of around 1,700 bases with only one open reading frame (ORF). The antigenomic ORF encodes the only protein of HDV, the Hepatitis Delta Antigen (HDAg) which is expressed in two isoforms: the small and the large HDAg (S- and L-HDAg); L-HDAg contains the S-HDAg

6.Expanded classification of Hepatitis D Viruses into subtypesHADI KARIMZADEH, ZAINAB USMAN, DMITRIJ FRISHMAN, MICHAEL ROGGENDORF

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and the 19 amino acid (aa) at the C-terminus.2 HDV is highly pathogenic and the current antiviral therapies with pegylated interferon have very low efficiency.3

To date, the strains of HDV isolated from patients have been classified into eight major genotypes/clades (HDV-1 to HDV-8).4 HDV-1 is the most prevalent genotype around the world including America, Europe, Middle East, East Asia, and Africa; the other genotypes are associated with distinct geographical and ethnic regions.5 Different HDV genotypes exhibit different clinical course and outcomes: HDV-1 strains show a broad spectrum of virulent and pathogenic phenotypes; HDV-2 and HDV-4 cause milder forms of liver disease; HDV-3 iso-lates are associated with outbreaks of fulminant hepatitis in South America.

It was thought that the HDV was only present in humans in invariable associa-tion with the HBV. However, in 2018 HDV-like sequences have been identified by metagenomic analyses in snakes (Boa constrictor),6 ducks (Anas species),7 rodents (Proechimys semispinosus) (Drosten, personal communication), fish, amphibians and invertebrates (termites).8 Most of these viruses have similar genomic fea-tures including size, circular and unbranched rod-like structures. The snake-de-rived HD protein bears 50% with the L-HDAg;6 the duck associated HDV protein shares only a homology of 32%.7

While the human HDV is detected solely in the liver because the HBsAg en-velope protein of the virus can bind only to hepatocytes, the HDV-like proteins are mostly expressed in tissue other than the liver and none is associated with another hepadnavirus infection. These sequences are transmissible, however the mechanism of transmission is not known. These exciting new findings are important to understand the origin of HDV and may be important to identify diseases of unknown origin.

Subtyping of HDV may be of importance for several reasons. Specific subtypes of HDV may have different response rates upon treatment with different drugs, as shown for other viruses like HCV; in chronic hepatitis C the rate of sustained viral responses with treatment by protease inhibitors was shown to be different for HCV subtype 1b vs 1a.9 In addition, the emergence of new HDV sequences presents an opportunity for understanding viral genome and evolution of new strains and subtyping of HDV which may be important to recognize shift with respect to mode of infection, or age in certain populations. For instance, relative to genotype 1 of HCV, there was a notable variation in the distribution of the prevalent subtypes 1a and 1b in different age groups; in individuals older than 50 years subtype 1b was most frequent and has been gradually substituted by subtype 1a over the last 20 years.10

Here, we describe comparative sequence analysis of the L-HDAg to identi-fy the plausible existence of specific geographic clusters (i.e. subtypes) within known HDV genotypes.

Expanded classification of Hepatitis D Viruses into subtypes 127

Elimination of redundant sequences of HDV isolates

In recent years, DNA sequencing technologies (i.e. next generation sequenc-ing [NGS] and Sanger sequencing) have resulted in enormous amount of data stored in public sequence repositories. To analyze these data poses a variety of challenges for the presence of multiple copies of highly similar sequences, which may produce misleading results due to the over-representation of identical se-quences. However, this problem can be solved by employing a sequence clus-tering method to establish a non-redundant dataset. Highly similar or identical sequences (i.e. nucleotide or protein) that appear as mere duplicates can be re-moved from the sequence data set using program known as CD-HIT (Cluster Da-tabase at High Identity with Tolerance)11 (Figure 6.1).

GenBankdatabase

Experimentaldata Public sequence

repository

CD-HIT

Redundantsequences

Non redundantsequences

Phylogeneticanalysis

Redundancy reduction by CD-HIT

Nucleotidedifference estimation

Non redundantdataset

Identification of new subtypes

MrBayes

Figure 6.1Workflow of sequence redundancy reduction and phylogenetic analysis.

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CD-HIT is a widely used program to reduce redundancy and to cluster and compare large sets of DNA and protein sequences. It takes a sequence file in FASTA format as an input and a user-defined similarity threshold (here a 90% identity is set as default). It outputs two files: a FASTA file of non-redundant se-quences and a text file of list of clusters. The resulting non-redundant sequences are representatives of a larger redundant dataset. In our recent study, we applied this approach to effectively remove highly identical sequences from our experi-mental dataset and sequence data from public nucleic acid sequence repository to perform a comprehensive evolutionary analysis of Hepatitis D Virus.12

Phylogenetic analysis for identification of HDV subtypes

To determine phylogenetic relationships of HDV genotypes and subtypes, an alignment is initially required. Sequence alignements of the L-HDAg compris-ing 645 bases, can be obtained from different multiple-sequence alignment pro-grams: ClustalO,13 MAFFT14 and MUSCLE.15 To study evolutionary relatedness among various group of organisms (species, populations) several phylogenetic methods have been widely used, e.g. MEGA, PHYLIP, PAUP, and MrBayes. In our recent study, we explored an alternative approach by cross-validating our findings using two different phylogenetic approaches, i.e. MEGA and MrBayes. Molecular Evolutionary Genetics Analysis (MEGA) estimates evolutionary distances using the Neighbor-joining (NJ),16 Maximum Likelihood (ML)17 and Maximum Parsimo-ny (MP) methods which are based on different nucleotide substitution models.18 The reliability and stability of phylogenetic tree analysis are assessed and assured by the bootstrap probability (at 1000 pseudoreplicates) for each interior branch of the tree. MrBayes program 3.2.6 software is a program for Bayesian inference using Markov chain Monte Carlo (MCMC) methods.19 MrBayes runs each align-ments for sufficient number of generations, e.g. 2x106 bootstrap replicates. The resulting consensus tree can be visualized using several tree viewer programs like TreeView, Dendroscope, Archaeopteryx and Figtree (v1.3.1) (Figure 6.2).

Although all the genotypes of HDV (HDV-1 to -8) were supported by a high bootstrap value of 100%, Delfino et al. summarized HDV classification into three HDV genogroups (genogroup 1=HDV-1, genogroup 3=HDV-3 and genogroup 2=all other genotypes, HDV-2,-4,-5 to -8).20 Recently, Le Gal5 and Miao et al.21 confirmed the original classification of HDV into eight major genotypes. Our findings were also in agreement with the previous literature confirming the rec-ognition of eight genotypes (i.e. HDV-1 to HDV-8).22

To further investigate the existence of subtypes within the known genotypes, a cluster of sequences (i.e. subtype) is often defined based on high bootstrap sup-port. For HDV, bootstraps ranging from 70% and up to 99% were used to distin-guish different clusters as subtypes within a genotype. Once the subtypes are


identified based on their high bootstrap support, pairwise genetic distances us-ing Kimura’s 2 parameter are computed.23 These pairwise genetic distances are used to calculate the average identity scores for each subtype and between two subtypes for all possible subtype combinations (e.g. subtype a-b, c, d, e, subtype b-c, d, e etc.). Based on our analysis we proposed that the genetic variability be-tween different genotypes was >10%, whereas the subtype showed genetic diver-gence of ≥3% to

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Amino acid signatures define HDV subtypes

Comparative protein sequence analysis is a powerful method to understand the evolutionary relationships among highly diverged sequences. In our analysis for HDV-1, we observed amino acid signatures at specific positions that distin-guishes one subtype from the other. These include amino acid positions 15, 95, 112, 117, 148, and 202 (Figure 6.3). It has been shown that the amino acid at po-sition 202 reflects a geographical background.5 These positions are also known as specificity determining positions (SDPs) and appear within the functional do-mains of L-HDAg, including RNA binding motifs (Ps 2-27), a coiled coil domain (Ps 31-52), a nuclear localization signal (Ps 66-88), a helix loop helix motif (Ps 111-138), PGRS proline/glycine-rich region and virus assembly signal or nuclear export signal (Ps 195-214).24 25

Geographical distribution of HDV is subtype specific

HDV genotype-1 (HDV-1) is the most prevalent variant of hepatitis D infec-tions; it spreads across different continents and countries including North Amer-ica, Africa, Europe, Mediterranean, Micronesia, Middle East, Central Asia, East Asia, Australia, and Russia.5 Interestingly, the HDV-1 subtypes identified in our study (supported by >70% bootstrap value)22 were classified as HDV-1a, -1b, -1c, -1d and -1e and were noted to be strongly associated with specific geograph-ic regions. Among these five subtypes, the two subtypes HDV-1a and HDV-1b consisted of sequences from sub-Saharan Africa, whereas the remaining three subtypes HDV-1c, HDV-1d and HDV-1e featured a distinct geographical distri-bution. HDV-1c was prevalent in Central Asia, HDV-1d was found in Middle East,

Table 6.1Comparison of nucleotide sequences among HDV-1 subtypes: (i) percent identity scores for each HDV-1 subtype (grey boxes) and (ii) inter-subtype sequence divergence scores (white boxes) for L-HDAg sequences.

HDV-1 Subtypes HDV-1a HDV-1b HDV-1c HDV-1d HDV-1e

Large Hepatitis Delta Antigen

(L-HDAg) nucleotide sequence

HDV-1a 94.78 7.08 4.65 3.85 5.75

HDV-1b 7.08 96.40 7.18 7.02 7.75

HDV-1c 4.65 7.18 97.41 3.00 3.03

HDV-1d 3.85 7.02 3.00 96.53 3.46

HDV-1e 5.75 7.75 3.03 3.46 96.76

Large Hepatitis Delta Antigen (L-HDAg) nucleotide sequence


Iran and Turkey, and HDV-1e was widely distributed across Europe. Meanwhile, Le Gal et al. in their recent study defined the classification of HDV-1 into four subtypes or subgenotypes (i.e HDV 1a-1e); in this study HDV-1a was mainly con-fined to central-eastern Africa; HDV-1b accounted for other parts of African con-tinent such as Guinea and Cote d’Ivoire; HDV-1c was constituted largely of Mi-croneasian isolates from Nauru and Kiribati, and lastly HDV-1d was comprised of isolates from Europe, North Africa, Central Asia, and North America.5

Additionally, it is not unusal to encounter highly diverged sequences which do not belong to any clade/genotype or subtype. Such unique sequences are of-ten referred to as outliers or borderline cases. However, these highly diverged sequences may in future be regarded as a new subtype or clade with the growing number of experimental data. In our analysis, we identified Micronesian isolates (Nauru and Kiribati) that might appear as potential new subtypes.

Future perspective of HDV classification: a consensus definition of genotypes and subtypes

Introducing the African isolates to the formerly described HDV types in 2006 resulted in 8 genotypes/clades.4 Despite further contribution of many groups to the number of HDV sequences during the last decade, this classification has not been updated since. To date, there are many open questions regarding the cor-relation between the HDV genotypes (subtypes) and different aspects of HDV infection including clinical symptoms and severity of the disease, response to therapy and the state of coinfection with HBV; parallel to our study of HDV sub-

HDV-1a

HDV-1b

HDV-1c

HDV-1d

HDV-1e

1 214

L-HDAg

15 95 112 117 148 202

Figure 6.3Subtype-specific amino acid residues predicted for HDV-1. The different color corresponds to differ-ent amino acid residues across HDV-1 subtypes.

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types, at least two other groups have recently identified possible HDV subtypes applying different approaches.20 21

Detailed characterization of HDV isolates along with the corresponding clin-ical information may help to address many of these issues. In order to facilitate the characterization of HDV genome as well as assisting scientist with fast pro-cessing of HDV sequences, we recently developed a comprehensive database of HDV genomes which can be reached at http://www.hdvdb.bio.wzw.tum.de/ (un-published data).

REFERENCES

1. Rizzetto M (2009). Hepatitis D: thirty years after. J Hepatol 50:1043-50.

2. Taylor JM (2012). Virology of hepatitis D virus. Semin Liver Dis 32:195-200.

3. Wedemeyer H, Yurdaydin C, Dalekos GN et al. (2011). Peginterferon plus adefovir versus either drug alone for hepatitis delta. N Engl J Med 364:322-31.

4. Le Gal F, Gault E, Ripault MP et al. (2006). Eighth major clade for hepatitis delta virus. Emerg Infect Dis 12:1447-50.

5. Le Gal F, Brichler S, Drugan T et al. (2017). Genetic diversity and worldwide distribution of the deltavirus genus: a study of 2,152 clinical strains. Hepatology 66:1826-41.

6. Hetzel U, Szirovicza L, Smura T et al. (2018). Identification of a novel deltavirus in boa constrictor. BioRxiv Preprint.

7. Wille M, Netter HJ, Littlejohn M et al. (2018). A divergent hepatitis D-like agent in birds. Viruses 10. pii: E720.

8. Chang W-S, Pettersson JH, Le Lay C et al. (2019). Novel hepatitis D-like agents in vertebrates and invertebrates. BioRxiv Preprint.

9. Hezode C, Fontaine H, Dorival C et al. (2014). Effectiveness of telaprevir or boceprevir in treatment-experienced patients with HCV genotype

1 infection and cirrhosis. Gastroenterology 147:132-42.

10. Ross RS, Viazov S, Renzing-Kohler K et al. (2000). Changes in the epidemiology of hepatitis C infection in Germany: shift in the predominance of hepatitis C subtypes. J Med Virol 60:122-5.

11. Li W, Godzik A (2006). Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658-9.

12. Benson DA, Clark K, Karsch-Mizrachi I et al. (2015) GenBank. Nucleic Acids Res 43(Database issue):D30-5.

13. Sievers F, Higgins DG (2014). Clustal Omega, accurate alignment of very large numbers of sequences. Methods Mol Biol 1079:105-16.

14. Katoh K, Misawa K, Kuma K et al. (2002). MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059-66.

15. Edgar RC (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792-7.

16. Saitou N, Nei M (1987). The neighbor-joining method: a new method for

reconstructing phylogenetic trees. Mol Biol Evol 4:406-25.

17. Guindon S, Gascuel O (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696-704.

18. Tamura K, Nei M, Kumar S (2004). Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 101:11030-5.

19. Ronquist F, Teslenko M, van der Mark P et al. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539-42.

20. Delfino CM, Cerrudo CS, Biglione M et al. (2018). A comprehensive bioinformatic analysis of hepatitis D virus full-length genomes. J Viral Hepat 25:860-9.

21. Miao Z, Zhang S, Ma Z et al. (2019). Recombinant identification, molecular classification and proposed reference genomes for hepatitis delta virus. J Viral Hepat 26:183-90.

22. Karimzadeh H, Usman Z, Frishman D et al. (2019). Genetic diversity of hepatitis D virus genotype 1 in Europe allows classification into subtypes. J Viral Hepat (in press).

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ABSTRACT

Carriers of the Hepatitis B Virus (HBV) superinfected with the Hepatitis D (Delta) Virus are at great risk of fast progression to liver cirrhosis and liver failure. Therapeutic options for these pa-tients are currently limited to pegylated interferon alpha with low response rates. Vaccination strategies tested in preclinical models induced specific B- and T-cell responses and protected from simultaneous infection, however failed to protect superinfection of chronic HBV carrier animals. Recently, new findings on innate and adaptive immune response in HDV infection became available which may help to develop more effective prophylactic and therapeutic vac-cines. These studies indicate: 1) in contrast to HBV, a strong innate immune response was ob-served in HDV infection; 2) patients with recovered HDV infection (HDV RNA negative) display robust HDV-specific CD8+ T cell responses, while these are more difficult to find in patients with chronic HDV infection; 3) the number of identified epitopes is still limited and epitopes may be primarily restricted by HLA-B alleles; 4) immune escape mutations within identified epitope occur frequently. New direct antiviral therapies using entry inhibitors, viral release in-hibitors or prenylation inhibitors are currently in phase II studies, which significantly reduce viral load. A better understanding of HDV specific immune responses in combination with new antiviral compounds may help to generate a protective or therapeutic vaccine against HDV.

Key words: Hepatitis B Virus, Hepatitis D (Delta) Virus, vaccines strategies, prophylactic and therapeutic vaccines, antiviral therapies, HDV specific immune responses, antiviral com-pounds.

Introduction

Hepatitis D (Delta) Virus (HDV) is an RNA virus which causes the most se-vere forms of chronic viral hepatitis in humans. HDV is a defective virus and needs Hepatitis B Virus (HBV) as a helper virus for its life cycle. It is estimated

17.New developments for prophylactic and therapeutic vaccines against HDVMICHAEL ROGGENDORF, HADI KARIMZADEH, STEPHANIE JUNG, CHRISTOPH NEUMANN-HAEFELIN

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that 15 to 20 million people are chronically infected with HDV worldwide. The strains of HDV isolated to date have been classified into eight genotypes (HDV-1 to HDV-8);1 HDV-1 is the most prevalent and geographically widespread strain, distributed across Europe, Middle East, East Asia, America and Africa. The other genotypes are associated with distinct geographical and ethnic regions: HDV-2 and HDV-4 are found in East Asia, HDV-3 isolates are exclusively found in South America (Amazon Basin of Brazil, Peru) and HDV-5 to HDV-8 prevails among the individuals from African origin. Deep phylogenetic analysis indicates sev-eral distinct HDV subtypes within different HDV genotypes, especially in HDV genotypes 1, 2 and 4, based on the analysis of the large hepatitis delta antigen (L-HDAg) region from the isolates studied so far. We have shown very recently that immune pressure on the HDV antigen has a great impact on viral evolution.2 It is thus very important to extend the nucleotide sequence assessed for geno-type analysis from the partial region, so called R0,3 to the whole L-HDAg region. Obviously, this analysis will be biased towards the genotypes which have been broadly studied, such as the genotype 1. Therefore, it would be necessary to col-lect more isolates form the region with less prevalent HDV genotypes, including Africa and South America. Detailed characterization of HDV subtypes may help to define the significance of HDV genome variabilities on clinical outcome and response to therapies.3 4

Two classical clinical pictures are described for HDV infection; simultaneous infection with both HBV/HDV and HDV superinfection of patients with a pre-ex-isting chronic HBV infection.5 Simultaneous infection is transient and self-limit-ing with >95% recovery rate in adults, and has the same characteristics as acute HBV mono-infection. A high titre of IgM anti-HBc is a key marker indicating si-multaneous HBV/HDV infection. In contrast, superinfection may lead to a severe acute phase of infection which, in most cases (80-90%), is followed by chronic HBV/HDV infection.

Apart from the above-mentioned two types of infections, simultaneous infec-tion and superinfection, there is a third pattern in which HDV infects hepato-cytes but cannot leave the cells simply because of absence of HBV and HBsAg. This was initially described as a possible event occurring in the transplantation setting6 7 and was confirmed experimentally in humanized mice in which HBV superinfection was able to rescue HDV production even after 6 weeks of HDV mono-infection,8 showing that HBV is not needed for HDV replication but only for packaging and viral release.9

The current therapeutic options for chronic HDV/HBV infection are limited; only alpha-interferon (IFN) is licensed for therapy of chronic hepatitis D.10 11 In early studies, it was shown that IFN is able to temporarily inhibit HDV replica-tion, but relapses may occur up to 1-2 years after discontinuation of therapy. IFN was replaced by pegylated interferon alpha-2a (Peg-IFN) which led to an in-

New developments for prophylactic and therapeutic vaccines against HDV 289

crease in the half-life and stability of the protein and to an improvement of the immunogenicity of this therapy.12

Soon after the discovery of the virus, studies on the pathogenesis of HDV in-fection and its replication properties were carried out in order to develop a vac-cine against HDV infection. For prevention of simultaneous HBV/HDV infection, no HDV-specific vaccine is needed as the available vaccines against HBV are very efficient to prevent also HDV infection. However, efforts to develop a vaccine to prevent superinfection of chronic HBV carriers failed, as summarized previous-ly.13 Some details of these studies will be described below. Recent findings on the innate and adaptive immune response may be used for developing more effec-tive vaccination strategies, which will be discussed in detail.

Based on the new insights into the innate and adaptive immune response, the main goal of this review is to describe prerequisites for the development of an effective vaccine to prevent HDV-superinfection of HBV carriers or design a ther-apeutic vaccine to cure chronic HBV/HDV infection. In addition, new antiviral compounds are considered which may be used in a combination with potential vaccine formulations.

Immunopathogenesis of HDV infection

In view of the satellite nature of the HDV and its obligatory association with the HBV to become infectious, immune responses against the HDV are always influenced by the immune response to the HBV coinfection. This leads to a more severe disease but also makes research on HDV-specific immunity more chal-lenging. Recent findings on innate and adaptive immune response are described below.

Innate immune response

The innate immune system detects pathogen-derived components by pat-tern recognition receptors (PRRs).14 Sensors of intracellular viral RNA are endo-somal toll like receptors (TLRs) as well as retinoic acid inducible gene I (RIG I) and melanoma differentiation associated gene 5 (MDA5) which belong to cyto-solic RIG I like receptors (RLRs). Activation of germline-encoded PRRs initiates divergent immune signalling pathways ultimately leading to pro-inflammatory cytokine and interferon (IFN) production which induce interferon-stimulated genes (ISGs) and antiviral state in both the infected and neighbouring cells. RIG I and MDA5 recognize distinct patterns of double-stranded RNA which leads to intramolecular conformational changes and allows their downstream interac-tion with mitochondrial antiviral signalling (MAVS) protein as a central adaptor molecule in RLR-dependent immune signalling.15

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Occurring invariably as a satellite virus to HBV under natural conditions, HDV research was hampered for a long time. Observed immunological effects were in-fluenced by the massive impact of HBV since first results could just be obtained in HBV/HDV coinfection. Of note, it was reported that HBV itself does not mount an IFN response but also does not block a response to additional inducers com-pletely.16 In contrast, HBV/HDV coinfection leads to a robust activation of the innate immune system.17 18 It was shown by Giersch at al. that humanized mice infected with both HBV and HDV exhibit enhanced ISG and cytokine expression levels compared to HBV-mono-infected or non-infected control animals.17 Also in different hepatoma cell lines, the pro-inflammatory cytokine response is en-hanced by expression of the large HDAg in an NF-kB dependent way.19 20

Within the last years, gain of knowledge was improved profoundly by new mod-el systems which allow investigating HDV in mono-infection only. Suárez-Am-arán et al. were the first to show that HDV-mediated interferon response is abol-ished and the number of HDV-producing cells is enhanced in MAVS-ko mice using adeno-associated HDV.21 Shortly after, knockdown and overexpression ex-periments in hepatoma-cell lines proved that ISG expression in response to HDV replication exclusively depends on MDA5 but not RIG I.22 Furthermore, UV-in-activated virus and viral protein are not sufficient for immune sensing, so active HDV replication is required for IFN induction.

Contradictory results exist regarding the influence of IFN on HDV replication which are most likely due to the fact that sensitivity of HDV to IFN treatment de-pends on the stage of infection. If IFN is administered during an early time point of HDV infection, viral replication is reduced in primary human hepatocytes and human hepatoma cell lines.22 23 Remarkably, this effect is abolished once the in-fection is established. This might be either due to HDV shielding from compart-mentalisation-dependent immune recognition by replicating in the nucleus24 or caused by impairment of IFN signalling by HDAg.25 Regardless of the mode of action, this fact complicates antiviral treatment.

In summary, it is beyond doubt that HDV leads to a robust activation of germline encoded PRRs and antiviral molecules. Also, it is well-known that in-nate immunity shapes the adaptive immune system by context-dependent anti-gen presentation, expression of costimulatory molecules and formation of a cy-tokine milieu affecting cells of adaptive immunity.26 However, it is left to further investigations how these effects can be used for specific manipulation of adap-tive immunity in therapeutic vaccinations against HDV.

Recent studies on the T cell response in HDV infection

Virus-specific immunity is thought to be essential for the elimination of hepa-totropic viral infections. It has been studied in detail in Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) infection.27 28 In these infections, a concerted action


of the different components of adaptive immunity was required for viral clear-ance: B cells produce neutralizing antibodies that inhibit viral spread, CD4+ T cells provide important help to B and CD8+ T cells, and CD8+ T cells serve as main effector cells that have direct cytolytic activity and produce antiviral cy-tokines for non-cytolytic virus control.

HDV-specific immunity, in contrast, was studied in little detail to date. Study of HDV-specific immunity is hampered by several aspects of HDV infection. 1. HDV infection is rare compared to HBV and HCV infection, especially in coun-

tries with high research resources such as the US or the European Union, with only 30 cases of HDV infections reported annually in Germany.

2. It remains so far elusive if adaptive immunity in HDV infection targets pri-marily L-/S-HDAg as the only protein encoded by the HDV genome, or rath-er HBsAg, an essential component of the HDV envelope encoded by the HBV genome. In addition, virus-specific immunity in simultaneous HBV/HDV infection of an HBV-naïve individual may strongly differ from virus-specific immunity in HDV superinfection of an HBV-positive host already affected by HBV-specific T cell exhaustion.

Regarding HDV-specific antibodies, Rizzetto et al. demonstrated as early as 1979 (two years after their first description of HDV) that HDV-specific antibodies are detectable only transiently and at low titers during acute-resolving infection, but are detectable at higher titers during persistent infection.29 It has thus been concluded that HDV-specific antibodies have no virus neutralizing capacity and are therefore not required for HDV clearance.

HDV-specific CD4+ T cells, in contrast, have not been studied until 1997, when Nisini et al. identified 4 HDV-specific CD4+ T cell epitopes that were, however, only targeted in inactive HDV carriers (normal ALT for >1 year, negative IgM anti-HD as marker of active liver disease).30 These findings resemble the lack of CD4+ T cell help in persistent HBV and HCV infection. In a follow-up study, the authors demonstrated that one of these four CD4+ T cell epitopes can be processed extracellularly, possibly enhancing presentation and targeting of this epitope.31 Interestingly, patients with persistent HBV/HDV infection display high frequencies of perforin-positive cytotoxic CD4+ T cells that are associated with markers of severe liver disease (high AST levels; low platelet counts).32 Howev-er the specificity of these cytotoxic CD4+ T cells (virus-specific versus bystand-er CD4+ T cells) has not been studied. In a very recent study by Landahl et al., HDV-specific CD4+ T cells were detectable in patients regardless of outcome of HDV infection (resolved versus persistent). These CD4+ T cells dominantly tar-geted two HDV-specific CD4+ T cell epitopes.33

The first two HDV-specific CD8+ T cell epitopes were described by Huang et al. in 2004.34 They are restricted by HLA-A*02 and were targeted in two patients with absent HDV replication (negative HDV RNA for >5 years, suggesting that

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these two patients had resolved HDV infection); however, CD8+ T cells specific for these two epitopes were not detectable by tetramer analysis in two patients with persistent HDV replication. In a follow-up study, the authors also modified the amino acid sequence of one of these two epitopes to achieve higher CD8+ T cell response induction and cytotoxicity at least in the mouse model.35

Possible mechanisms of T cell failure in persistent HDV infection were in-vestigated in a recent study by Schirdewahn et al.36 When comparing the bulk T cell phenotype in patients with HBV/HDV infection versus HBV mono-infec-tion or healthy controls, HDV-infected patients displayed higher expression of the senescence marker CD57 on CD8+ T cells. Upon stimulation with overlap-ping peptides spanning the L-HDAg protein, the authors observed only weak (HDV-specific) T cell proliferation and cytokine production. Addition of the pro-inflammatory third signal cytokine IL-12 increased CD4+ and CD8+ T cell responses, while blockade of the PD-1 or CTLA-4 pathways had a minor effect. These data further support the absence and/or exhaustion of HDV-specific T cells in persistent infection and indicate that a characterization of T cell exhaus-tion and dysfunction on a cellular level using HLA tetramers will be important.

Another candidate mechanism of virus-specific CD8+ T cell failure is mutation-al viral escape, a mechanism that has been well defined in HIV and HCV infec-tion.37 Indeed, viral sequence mutations in candidate CD8+ T cell epitopes have been described in patients with persistent HDV infection in two studies.38 39 How-ever, the authors did not perform functional analyses to confirm that the observed mutations indeed mediate viral escape from virus-specific CD8+ T cell responses.

Due to the lack of known HDV-specific CD8+ T cell epitopes that can be used for further studies on HDV-specific CD8+ T cell failure, and to the lack of ex-perimental evidence for viral escape from HDV-specific CD8+ T cell responses, we recently set out to identify L-HDAg-specific CD8+ T cell epitopes restricted by different HLA class I alleles and to define the role of viral escape from these HDV-specific CD8+ T cell responses.2 Strikingly, when we performed in silico prediction as well as in vitro HLA affinity analyses to identify L-HDAg-derived peptides that bind to common HLA class I alleles, we failed to identify binders to HLA-A*01, A*02, A*03, A*24, and B*07 (compare Table 17.1: few HDV-specific binders were predicted in silico but failed to bind in vitro). In contrast, when we tested for binders to HLA-B*27, an HLA type that is rather rare (approx. 5-8%) in most populations world-wide and that has been described to be protective in HIV and HCV infection,40 we identified two optimal HLA-B*27 binding peptides. These two peptides were then confirmed to correspond to two novel HDV-spe-cific CD8+ T cell epitopes targeted in patients with resolved HDV infection. Overall, as compared to other nucleoproteins like HBV core or HCV core protein which have a similar length of amino acids, a much lower number of epitopes could be predicted.2 These results indicate that HDV may have evolved to es-cape from immune recognition restricted by common HLA class I alleles, while


it is still recognized in the context of rare HLA class I alleles such as HLA-B*27. The two novel HLA-B*27-restricted CD8 T cell epitopes are located in a relatively conserved region of L-HDAg. However, when we performed viral sequence anal-yses in 8 HLA-B*27-positive versus 96 HLA-B*27-negative patients with chron-ic HDV infection, we detected molecular footprints within these epitopes in HLA-B*27-positive patients. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infection, indicating that these substitutions represent viral escape mutations. In agreement with previous stud-ies, HLA-B*27+ patients with chronic HDV infection did not display CD8+ T cell responses to the two HLA-B*27-restricted CD8+ T cell epitopes. Indeed, even af-ter applying an ultra-sensitive tetramer-based enrichment strategy, no HDV-spe-cific CD8+ T cells could be detected in patients with chronic HDV infection.

Two studies by the group of Barbara Rehermann and our group further analyz-ed HDV-specific CD8+ T cells in patients with resolved or chronic HDV infection.

Table 17.1Comparison of predicted epitopes for HBV core, HCV core, and L-HDAg from HDV

HCV HBV HDV

Viral properties Size of the virion 45-65

Genome length 9500 3200 1700

Number of ORFs 1 4 1

Total amino acids 3030 1584 196-214

Size of core protein 191 185 214

Epitope prediction A*01 0 1 0

A*02 11 7 2

A*03 6 1 0

A*11 5 3 1

A*24 1 2 0

B*07 7 4 2

B*15 7 8 2

B*18 1 4 0

B*27 4 9 2

B*35 4 8 4

Sum 46 47 13

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Indeed, Kefalakes et al. used overlapping peptides to identify 6 novel HDV-spe-cific CD8+ T cell epitopes restricted by 5 different HLA-B alleles. In patients with chronic HDV infection, HDV-specific CD8+ T cells were detectable by peptide/HLA class I tetramers, however, they were either functionally constrained or tar-geted epitopes with escape mutations.41

Karimzadeh et al. demonstrated that viral escape from HDV-specific CD8+ T cells is only observed for epitopes restricted by relatively infrequent HLA class I alleles, indicating that HDV has already adapted at the population level to avoid recognition by frequent HLA class I alleles.42 This study also identified 5 novel HDV-specific CD8+ T cell epitopes; interestingly, two epitopes overlapped be-tween these two recent studies.

In summary, previous studies as well as very recent studies indicate that HDV-specific CD4+ and CD8+ T cell are present in acute-resolving HDV infection (Table 17.2); in persistent HDV infection, however, HDV-specific CD8+ T cells are either completely deleted or are in a heavily dysfunctional state. In addition, viral

Table 17.2HDV-specific CD8+ T cell epitopes identified to date

HLA allele Position in L-HDAg AA sequence Reference

26-34 A*02:01 KLEDLERDL Huang et al. 200434

43-51 A*02:01 KLEDENPWL Huang et al. 200434

46-54 B*18:01B*44:02/:03

DENPWLGN Karimzadeh et al. 201942

Kefalakes et al. 201941

81-90 B*37:01 VDSGPRKRPL Karimzadeh et al. 201942

B*37:01 100-108 QDHRRRKAL Karimzadeh et al. 201942

99-108 B*27:05/:02 RRDHRRRKAL Karimzadeh et al. 20182

104-112 B*27:05/:02 RRKALENKK/R Karimzadeh et al. 20182 Kefalakes et al. 201941

140-149 B*41:01 RERRVAGPPV Karimzadeh et al. 201942

170-179 B*15:01 SMQGVPESPF Karimzadeh et al. 201942

189-196 B*58:01 RGSQGFPW Kefalakes et al. 201941

192-200 B*35:01*B*52:01

QGFPWDILF Kefalakes et al. 201941

194-202 B*07:02/:05#

B*35:01FPWDILFPA Kefalakes et al. 201941

*B*35:01 also presents the 8-mer L-HDAg193-200 (GFPWDILF).#B*07:02/:05 also present the 10-mer L-HDAg193-202 (GFPWDILFPA).


escape may contribute to failure of HDV-specific CD8+ T cells in persistent infec-tion. Of note, in contrast to nuclear proteins of HBV or HCV, a very low number of T cell epitopes has been predicted for HDV, which might make it much more difficult for a vaccine to induce an effective multi-specific T cell response (Table 17.1). Future research is needed to broaden the repertoire of described HDV-spe-cific CD4+ and CD8+ T cell epitopes, characterize the mechanisms of T cell fail-ure in more detail, define the relative roles of HDV- and HBV-specific T cells in simultaneous versus superinfection and establish strategies to restore antiviral immune efficacy in chronic infection.

New antiviral compounds which reduce viral load and may be used in combination with new vaccines

The immune system uses the concept of immune tolerance to limit an immune reaction and prevent damage of vital organs. The consequence of this mechanism is a silenced immune response due to high loads of viral antigens during persistent exposure. Specifically the liver uses unique immune modulatory functions pro-moting immune tolerance rather than effector responses to viral antigens.

Infections with several non-cytopathic viruses such as lymphocytic chori-omeningitis virus (LCMV) or HBV are associated with overwhelming antigenic viral loads.43 Likewise, infection with HDV may also be accompanied by a high viral load, up to 109-1010 particles per ml of blood,which could represent an im-mune evasive strategy of HDV to prevent T-cell mediated clearance of the virus and to establish chronic infection. For re-constituting an effective HDV-specific T-cell response, it might be necessary to first reduce the viral antigen burden. A long-term suppression of HDV proteins may allow for the generation of func-tional, antigen-specific T cells or the restoration of T-cell function by additional vaccination. There are currently three compounds investigated in clinical trials, which have been shown to reduce viral load.

Entry inhibitor myrcludex B

By their mode-of-action, novel HBV entry inhibitors (e.g. myrcludex B=M)44 45 cannot prevent transcription, antigen expression nor release of HBV/HDV pro-teins. However they have the potential to efficiently prevent re-infection and block de novo infections of HBV naïve hepatocytes or of the new cells arising dur-ing hepatocyte turnover. Therefore HBV/HDV entry may contribute to reduce the number of infected cells and accelerate the process of virus elimination.46 Myrcludex B could even contribute to the induction of HBV preS-specific an-tibody responses and may be considered for concomitant administration with a therapeutic vaccination schedule against chronic hepatitis B/D in the future.

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Release inhibitor/Nucleic acid polymer (NAP) REP 2139

Nucleic acid polymers (NAPs) are single stranded phosphorothioated oli-gonucleotides (PS-ONs), which have a sequence-independent unique ability to block the secretion of HBsAg from HBV-infected hepatocyte.47 48 However, the mechanism which inhibits HBsAg-release from hepatocytes is not yet un-derstood. So far, no significant innate immune response, like cell-intrinsic im-munity, inflammatory or antiviral responses was detected in isolated primary human hepatocytes, Kupffer cells and liver sinusoidal endothelial cells using a variety of NAPs including REP 2139.49 Recently, they have been evaluated as a therapeutic agent for the treatment of chronic HBV infection50 and HBV/HDV coinfection.51

The first open-label, non-randomized study (REP 102) examined REP 2139 in monotherapy of 12 patients with HBeAg positive chronic hepatitis B.50 NAP monotherapy was accompanied by a 2-7 log reduction of serum HBsAg, a 3-9 log reduction in serum HBV DNA and the appearance of serum anti-HBs (10-1712 mIU/ml). Control of HBV infection persisted after removal of all therapies in eight of nine patients receiving combination therapy with Peg-IFN and per-sisted for more than two years in four of these patients. In the subsequent REP 301 study the safety and efficacy of REP 2139 in combination with Peg-IFN was assessed in twelve patients with chronic HBV/HDV coinfection.51 Most patients showed a reduction of HBsAg by 2-7 logs and HDV RNA became undetectable in eleven of twelve patients during therapy. One year after the end of therapy, five patients were still negative for HBsAg, five had high titres of anti-HBs and seven patients had no detectable HDV RNA in the blood indicating functional control of HBV and HDV coinfection.

Inhibition of the farnesylation of the L-HDAg by lonafarnib

The farnesylation (prenylation) of the large HD antigen is required for virion assembly by interaction with HBsAg. The inhibition of prenylation with lona-farnib (LNF) resulted in the reduction of the load of HDV in serum. In a proof-of-concept study, oral LNF decreased HDV RNA during 4 weeks of treatment. Higher LNF monotherapy doses led to greater decreases in HDV viral load than achieved in the original proof-of-concept study. However, this was associated with increased gastrointestinal adverse events.52 53

In conclusion, the combination of antiviral therapy with either myrcludex B, NAPs or LNF with a therapeutic vaccine might be beneficial for patients with chronic HBV/HDV coinfection.


Preventive and therapeutic vaccines against HDV in preclinical models

In recent years several efforts have been undertaken to develop a prophylactic vaccine against HDV infection in order to prevent HDV superinfection of chron-ic HBsAg carriers. The existing vaccines against HBV protect HBV-naïve subjects from HDV coinfection by inducing antibodies to the common envelope of both viruses. To date only preclinical studies on prophylactic HDV vaccines have been performed, which were reviewed previously.13 54

Because antibodies to the p24 and p27 proteins of HDV do not neutralize the virus and the viral particle is covered by a coat of HBsAg against which carriers of the HBV are unable to elicit an antibody response, classical vaccines which induce neutralizing antibodies cannot be expected to prevent superinfection of chronic HBV carriers with HDV.

It was shown that vaccines which induce virus-specific T cells were able to prevent infection by suppression of replication, e.g., by cytokine secretion. In a second step, these virus-specific T cells are able to eliminate infected cells by their cytolytic activity and thus prevent the spread of the virus,55-58 while T cell vaccines cannot provide sterile immunity, because they do not prevent infection of target cells. However, they may nevertheless eliminate infected cells by stop-ping with their cytotoxic activity the spread of the virus at a very early phase of infection. The induction of such cytotoxic HDV specific CD8 T cells by a vaccine was first described in a mouse model.59 This vaccine prototype has been later evaluated in woodchucks to prevent simultaneous infection by the Woodchuck Hepatitis Virus (WHV) and the HDV, as well as superinfection of animals which were WHV carriers. The T cell vaccine induced a vigorous HDV-specific T cell re-sponse which is a requirement to prevent the spread of the virus.

Preventive vaccine

A T cell vaccine with DNA prime and adenoviral vectors boost immunization regimen induced an HDV specific CD8 T cell response and prevented HDV infec-tion after simultaneous challenge with WHV and woodchuck adapted HDV. The vaccine-induced specific CD8 T cell response was effective in preventing HDV rep-lication and spread in the liver.54 However, HDV vaccines were much less promis-ing in preventing superinfection; the T-cell responses induced by the current DNA prime and viral vector boost immunization were insufficient to prevent the spread of HDV in chronic WHV carrier animals.54 60 61

Little progress was achieved to improve prophylactic HDV vaccines for HBsAg carriers over the last 5 years, due primarily to insufficient knowledge on the in-nate and T cell immune response to HDV. New information concerning innate and adaptive T cell responses, optimized prime boost regiments and the avail-

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ability of new mouse models with a persistent HDV replication, may promote preclinical studies for a prophylactic vaccine.

The vectors used for prime boost vaccination have to be checked whether the amino acid sequence of the vaccine contains the complete open reading frame (ORF) of the L-HDAg with the appropriate known epitopes. The new MVA vector for the boost vaccination has the advantage that most of the patients to be vac-cinated against HDV have no immunity against this virus. The woodchuck has been established as a very good model for studies on hepadna viral and HDV in-fections, though the number of tools to monitor virus specific immune respons-es is limited. There are new mouse models which use AAV vectors to establish a chronic HDV/HBV infection, that may be helpful to test different prime boost regiments and the persistence of the primed virus specific T cells.

There are however major problems over the long run. It is well known from vi-ral infections and vaccination studies that after the elimination of the infectious agent, the generated virus specific T cells undergo a retraction phase which results in persistence of only a small number of virus specific memory T cells; in addition, in chronic HBV patients superinfected with HDV the virus spread is very fast and therefore the amplification of virus specific T cells may come too late to prevent infection and spread of the virus. As a consequence, HBsAg carriers exposed to HDV would need a continuous revaccination to keep HDV specific T cells at suffi-cient levels; given that this treatment would be quite expensive and made difficult by logistic problems, only high-risk patients could be considered for vaccination.

Perspective for the development of a therapeutic vaccine to treat chronically HBV/HDV infected patients

The perspectives to develop therapeutic vaccines for HBV have lately in-creased. In preclinical HBV models a combination of antiviral treatment to re-duce viral load and prime boost vaccination is needed to achieve a proper T cells response and virus neutralizing antibodies.55 57 62 63 This information may be used to develop a therapeutic HDV vaccine in chronic HDV/HBV infection. Though the response to Peg-IFN is low and so far no other therapy has been licensed, the new antiviral drugs against HDV currently in clinical studies may be helpful to reduce HDV replication by inhibiting the release or the assembly of the virus, thus increasing the efficacy of vaccine. The optimal therapeutic vaccination protocol for a phase I study would be priming with a DNA vaccine containing L-HDAg or a protein vaccine with L-HDAg+ adjuvant and subse-quent boost with MVA or adenoviral vectors (AD5 or AD35) (Figure 17.1). The optimal patients for such a study should be negative for HBV DNA, serocon-verter from HBeAg to anti-HBe, should have low concentrations of HBsAg and without previous IFN treatment. For the design and perspectives of therapeutic vaccines in the future, recent knowledge concerning HDV-specific CD8+ T cell


epitopes may be used to develop a multi epitope vaccine. This type of vaccine may become available with the recently described CD8+ T cell epitopes.41 42 However, a major problem is the high variability of HDV proteins64 and the im-mune escape within epitopes is a challenging task.

Conclusions

The recent findings regarding HDV-specific immunity will be beneficial for the development of a protective as well as therapeutic vaccine in several aspects. 1. The finding that HDV has well adapted to hide from recognition by common

HLA class I alleles points out that prophylactic as well as therapeutic vaccines will need to cover CD8+ T cell epitopes restricted by less common HLA class I alleles. Thus, use of HLA-A*02 as a common experimental background to study vaccine-induced immune responses will not be feasible. Rather, addi-tional efforts are needed to identify HDV-specific CD8+ T cell epitopes restrict-ed by rare HLA class I alleles in addition to HLA-B*27.

2. The severe exhaustion/deletion of HDV-specific CD8+ T cells in persistent HDV infection suggests that therapeutic vaccination as an antiviral approach in chronic HDV infection might require immunomodulation by several mecha-nisms, e.g. blockade of PD-1 and/or CTLA-4 in addition to application of pro-in-flammatory stimuli. Of course, such heavy immunomodulatory interventions will only be suitable for a short duration and under close safety monitoring.

3. Viral escape as an additional mechanism of T cell failure needs to be consid-ered especially in the setting of therapeutic vaccination, since boostering of virus-specific CD8+ T cell responses that target mutated epitopes has shown to be of no clinical benefit in HCV infection.65

Months

Antiviral treatment

MVA or AD5 boostControl viral load DNA/Protein prime

0 3 6

Figure 17.1Scheme of therapeutic vaccination in chronic HDV/HBV infection.

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Summer Academy “Infection and Immunity in times of COVID ...€¦ · the evolutionary relationships among highly diverged sequences. In our analysis for HDV-1, we observed amino