Prior flavivirus immunity skews the yellow fever vaccine response to cross-reactive antibodies with potential to enhance dengue virus infection.

The yellow fever 17D vaccine (YF17D) is highly effective but is frequently administered to individuals with pre-existing cross-reactive immunity, potentially impacting their immune responses. Here, we investigate the impact of pre-existing flavivirus immunity induced by the tick-borne encephalitis virus (TBEV) vaccine on the response to YF17D vaccination in 250 individuals up to 28 days post-vaccination (pv) and 22 individuals sampled one-year pv. Our findings indicate that previous TBEV vaccination does not affect the early IgM-driven neutralizing response to YF17D. However, pre-vaccination sera enhance YF17D virus infection in vitro via antibody-dependent enhancement (ADE). Following YF17D vaccination, TBEV-pre-vaccinated individuals develop high amounts of cross-reactive IgG antibodies with poor neutralizing capacity. In contrast, TBEV-unvaccinated individuals elicit a non-cross-reacting neutralizing response. Using YF17D envelope protein mutants displaying different epitopes, we identify quaternary dimeric epitopes as the primary target of neutralizing antibodies. Additionally, TBEV-pre-vaccination skews the IgG response towards the pan-flavivirus fusion loop epitope (FLE), capable of mediating ADE of dengue and Zika virus infections in vitro. Together, we propose that YF17D vaccination conceals the FLE in individuals without prior flavivirus exposure but favors a cross-reactive IgG response in TBEV-pre-vaccinated recipients directed to the FLE with potential to enhance dengue virus infection.

Distinct and dynamic activation profiles of circulating dendritic cells and monocytes in mild COVID-19 and after yellow fever vaccination.

Dysregulation of the myeloid cell compartment is a feature of severe disease in hospitalized COVID-19 patients. Here, we investigated the response of circulating dendritic cell (DC) and monocyte subpopulations in SARS-CoV-2 infected outpatients with mild disease and compared it to the response of healthy individuals to yellow fever vaccine virus YF17D as a model of a well-coordinated response to viral infection. In SARS-CoV-2-infected outpatients circulating DCs were persistently reduced for several weeks whereas after YF17D vaccination DC numbers were decreased temporarily and rapidly replenished by increased proliferation until 14 days after vaccination. The majority of COVID-19 outpatients showed high expression of CD86 and PD-L1 in monocytes and DCs early on, resembling the dynamic after YF17D vaccination. In a subgroup of patients, low CD86 and high PD-L1 expression were detected in monocytes and DCs coinciding with symptoms, higher age, and lower lymphocyte counts. This phenotype was similar to that observed in severely ill COVID-19 patients, but less pronounced. Thus, prolonged reduction and dysregulated activation of blood DCs and monocytes were seen in a subgroup of symptomatic non-hospitalized COVID-19 patients while a transient coordinated activation was characteristic for the majority of patients with mild COVID-19 and the response to YF17D vaccination.

Single Nucleotide Polymorphism rs6445975 in the PXK Gene Is Correlated with Susceptibility and Clinical Characteristics of Systemic Lupus Erythematosus.

Background: Recently, genome-wide association studies (GWAS) have discovered several single nucleotide polymorphisms (SNPs) and loci associated with the risk of systemic lupus erythematosus (SLE). rs6445975 (T>G; intronic variant) polymorphism in the PXK gene is one of these loci. However, there was an inconsistency between the results of replicative studies on European and Asia ancestry. This study aimed to assess the possible association between rs6445975 polymorphism with SLE risk in the Iranian population. Methods: Genotype and allele distribution of rs6445975 polymorphism were investigated in 110 patients with SLE and 115 healthy controls in Isfahan University of Medical Sciences, Isfahan, Iran in 2019 via real-time PCR high resolution melting method (HRM). Results: GG and TG genotypes, but not TT genotype, were associated with increased risk of SLE (GG vs TT; OR= 7.538; 95%CI [3.47, 17.066] and TG vs TT; OR=2.21; 95%CI [1.06, 4.72]). Inheritance analysis revealed that TG + GG was correlated with the increased risk of SLE disease in the dominant model (OR=3.928; 95%CI [2.056, 7.74]). Moreover, subjects with the G allele were more frequently affected with SLE than individuals with the T allele (OR= 3.55; 95%CI [2.37, 5.36]). The G allele in patients was correlated with serum concentration of CRP, ESR, anti-dsDNA antibody, C3, and C4 and presentation of some clinical manifestations such as kidney involvements and skin lesions (P<0.05). Conclusion: Our findings suggest a substantial association between rs6445975 polymorphism in the PXK gene with susceptibility and clinical characteristics of SLE in the Iranian population.

Impact of Single Nucleotide Polymorphism in the ANKRD55 Gene on Occurrence and Clinical Characteristics of Rheumatoid Arthritis.

Background: Rheumatoid Arthritis (RA) has multifactorial etiology and numerous genetic and environmental factors have been related to an increased risk of RA. Recently, Genome-Wide Association Studies (GWAS) suggested a large number of Single Nucleotide Polymorphisms (SNPs) loci affecting the susceptibility to RA. One of these loci is rs6859219 (C>A), a functional polymorphism in the ANKRD55 gene which was associated with the expression of ANKRD55 and IL6ST. In the current study, we evaluated the possible association between rs6859219 (intronic variant) in the ANKRD55 gene with RA risk in the Iranian population. Methods: A case-control study using 118 RA patients and 115 healthy counterparts was undertaken in order to determine rs6859219 genotypes using real-time polymerase chain reaction High-Resolution Melting (HRM) method. Results: There was a significant difference in the genotype and allele frequencies of rs6859219 between patients and controls (p<0.001). Logistic regression analysis demonstrates that CC genotype and C allele increased the risk of RA (OR for CC genotype= 7.12; 95%CI [3.51-15.05]/OR for C allele=4.16; 95%CI [2.78-6.28]). Furthermore, regarding the dominant and recessive model of inheritance, RA patients indicated obvious association of the rs6859219 variant compared to healthy controls (p<0.001). Moreover, in the patient group, there was a significant correlation between C-Reactive Protein (CRP) concentration with rs6859219 polymorphism (p<0.001). Conclusion: Our findings propose a substantial correlation between rs6859219 polymorphism and RA risk and clinical characteristics of this disease in the Iranian population.

Efficacy and safety of the biosimilar denosumab candidate (Arylia) compared to the reference product (Prolia®) in postmenopausal osteoporosis: a phase III, randomized, two-armed, double-blind, parallel, active-controlled, and noninferiority clinical trial.

BACKGROUND/OBJECTIVE: Osteoporosis is a global health concern with an increasing prevalence worldwide. Denosumab is an antiresoptive agent that has been demonstrated to be effective and safe in osteoporotic patients. This study aimed to compare the efficacy and safety of the biosimilar denosumab candidate (Arylia) to the originator product (Prolia®) in postmenopausal osteoporotic patients. METHODS: In this randomized, double-blind, active-controlled, noninferiority trial, postmenopausal osteoporotic patients received 60 mg of subcutaneous Arylia or Prolia® at months 0, 6, and 12 and were followed up for 18 months. The primary endpoint was the noninferiority of the biosimilar product to the reference product in the percentage change of bone mineral density (BMD) in 18 months at the lumbar spine (L1-L4), total hip, and femoral neck. The secondary endpoints were safety assessment, the incidence of new vertebral fractures, and the trend of bone turnover markers (BTMs). RESULTS: A total of 190 patients were randomized to receive either biosimilar (n = 95) or reference (n = 95) denosumab. In the per-protocol (PP) analysis, the lower limits of the 95% two-sided confidence intervals of the difference between Arylia and Prolia® in increasing BMD were greater than the predetermined noninferiority margin of - 1.78 at the lumbar spine, total hip, and femoral neck sites (mean differences [95% CIs] of 0.39 [- 1.34 to 2.11], 0.04 [- 1.61 to 1.69], and 0.41 [- 1.58 to 2.40], respectively). The two products were also comparable in terms of safety, new vertebral fractures, and trend of BTMs. CONCLUSION: The efficacy of the biosimilar denosumab was shown to be noninferior to that of the reference denosumab, with a comparable safety profile at 18 months. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03293108 ; Registration date: 2017-09-19.

A High-Throughput Yellow Fever Neutralization Assay.

Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks for surveillance and to evaluate vaccine efficacy in population-wide studies. All of this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming, and limited in throughput, the classical plaque reduction neutralization test (PRNT) is still considered the gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here, we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards, showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika, and dengue viruses amenable to differential diagnostics. IMPORTANCE Fast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance, and monitoring of vaccine efficacy. Although classical PRNT remains the gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as low throughput and overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing a large number of samples in a highly standardized manner and has the potential to be implemented for clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika, and dengue viruses, opening new avenues for differential diagnostics.

Single nucleotide polymorphism rs5029937 in TNFAIP3 gene is correlated with risk of rheumatoid arthritis.

Background: Rheumatoid arthritis (RA) is a progressive and common autoimmune disease with multifactorial etiology. Several pieces of research show that genetic factors play a major role in the incidence of RA. Several genome-wide association studies (GWAS) have identified the tumor necrosis factor alpha inducible protein 3 (TNFAIP3) genes as one of the candidate loci. The TNFAIP3 gene encoding ubiquitin-editing protein A20 witch restricts B cell survival and prevents autoimmunity. Previous studies have indicated that single nucleotide polymorphisms (SNPs) in the TNFAIP3 gene are correlated with several autoimmune disorders. In the present study, we assessed the possible association between SNP rs5029937 (intronic variant) in the TNFAIP3 gene with RA risk in the Iranian population. Methods: A case-control study using 50 RA patients and 50 control subjects was undertaken to evaluate rs5029937 (G>T) genotypes using real-time PCR high resolution melting method (HRM). The SPSS22 was used for statistical analyses and the significance level was set at P<0.05. Results: Logistic regression analysis demonstrates that homozygous TT + heterozygous TG genotypes compared with GG genotype increase the risk of RA (TT+TG vs GG; P= 0.004, OR= 3.46; 95%CI [1.492-8.075]). Also, individuals with allele T were more frequently affected with RA than subjects with G allele (T vs G; P= 0.004, OR= 2.61; 95%CI [1.382-4.919]). Conclusion: Our findings propose a substantial correlation between rs5029937 (G>T) polymorphism and RA risk in Iranian population.

Defective Interfering Genomes and the Full-Length Viral Genome Trigger RIG-I After Infection With Vesicular Stomatitis Virus in a Replication Dependent Manner.

Replication competent vesicular stomatitis virus (VSV) is the basis of a vaccine against Ebola and VSV strains are developed as oncolytic viruses. Both functions depend on the ability of VSV to induce adequate amounts of interferon-α/ß. It is therefore important to understand how VSV triggers interferon responses. VSV activates innate immunity via retinoic acid-inducible gene I (RIG-I), a sensor for viral RNA. Our results show that VSV needs to replicate for a robust interferon response. Analysis of RIG-I-associated RNA identified a copy-back defective-interfering (DI) genome and full-length viral genomes as main trigger of RIG-I. VSV stocks depleted of DI genomes lost most of their interferon-stimulating activity. The remaining full-length genome and leader-N-read-through sequences, however, still triggered RIG-I. Awareness for DI genomes as trigger of innate immune responses will help to standardize DI genome content and to purposefully deplete or use DI genomes as natural adjuvants in VSV-based therapeutics.

Assessment of Treatment Safety and Quality of Life in Patients Receiving Etanercept Biosimilar for Autoimmune Arthritis (ASQA): A Multicenter Post-marketing Surveillance Study.

INTRODUCTION: Phase IV post-marketing surveillance studies are needed to evaluate the real-world safety and effectiveness of drug products. This study aimed to evaluate the safety and effectiveness of biosimilar etanercept (Altebrel, AryoGen Co., Iran) in patients with rheumatoid arthritis (RA), ankylosing spondylitis (AS), and psoriatic arthritis (PsA). METHODS: In this open-label, multicenter, prospective, observational, post-marketing surveillance study, 583 patients received biosimilar etanercept 25 mg twice weekly or 50 mg once weekly and were followed up to 12 months. The primary objective was to evaluate the safety of biosimilar etanercept by documenting all the adverse events in the case report forms throughout the study period. The secondary objective was to evaluate the effectiveness of biosimilar etanercept in study patients, where longitudinal changes in health assessment questionnaire (HAQ), pain, and disease activity scores were assessed. RESULTS: A total of 583 patients (44.80 ± 13.09 years of age) were included and followed for an average of 8.12 ± 3.96 months. Among all patients, 172 (29.50%) experienced at least one adverse event, and injection site reaction, abdominal pain, and upper respiratory tract infection were the most common. HAQ scores decreased from 1.32 ± 0.77 at baseline to 0.81 ± 0.61 at 12 months in patients with RA/PsA (p < 0.01) and from 0.82 ± 0.58 at baseline to 0.66 ± 0.63 at 12 months in patients with AS (p = 0.18). Pain scores decreased from 6.49 ± 2.41 at baseline to 3.51 ± 2.39 at 12 months (p < 0.01). CONCLUSION: The results demonstrated the real-world safety and effectiveness of biosimilar etanercept in patients with RA, PsA, and AS. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT04582084.

Reply to the Letter of Charre et al. "Mis-Genotyping of Some Hepatitis D Virus Genotype 2 and 5 Sequences Using HDVdb".

We thank Charre and colleagues for spotting the mis-annotation of sequences in our database, which was caused by human error [...].

Efficacy of synbiotic supplementation in improving rheumatoid arthritis.

BACKGROUND AND PURPOSE: Today, improving rheumatoid arthritis (RA) as a chronic inflammatory disease is attributed to the proper status of the gut microbiota. Although some supplements containing beneficial live microorganisms (probiotics) can reduce inflammation by altering the bacterial composition of the gut, there is limited information on the effect of synbiotic (probiotics mixed with prebiotics) supplements on RA. Therefore, this study aimed to evaluate the anti-inflammatory effects of a synbiotic supplement as an adjuvant therapy in rheumatic patients. Moreover, for the first time, it was attempted to investigate whether addition of a synbiotic (1000 mg/day) to the combination of methotrexate and prednisolone increases the effectiveness of these antirheumatic drugs. EXPERIMENTAL APPROACH: Eligible patients (186 subjects) were randomly divided into two groups. Both groups received their standard routine antirheumatic drugs, methotrexate and prednisolone. Moreover, the first group received a daily oral synbiotic supplement (1000 mg) for 3 months while the second group received a placebo. Various parameters indicating RA status were evaluated at baseline (time 0) and 3 months after the treatment. FINDINGS / RESULTS: The results showed the changes in the level of RA indicators, including tender joint count with a range of 0 to 28 joints, swollen joint count with a range of 0 to 28 joints, visual analog scale, erythrocyte sedimentation rate, CRP, and disease activity score based on 28 joints, after 3 months. CONCLUSION AND IMPLICATIONS: Overall, no significant differences in the measured parameters were observed between synbiotic and placebo groups probably due to the short duration of the treatment period, and it is suggested to extend the treatment period to six months.

C1QTNF4 gene p.His198Gln mutation is correlated with early-onset systemic lupus erythematosus in Iranian patients.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease with multifactorial etiology. Several studies show that genetic factors have an important part in the incidence of SLE. The C1QTNF4 gene is involved in the regulation of the inflammatory pathways by pro-inflammatory function. In the present study, we have evaluated the association between C1QTNF4 gene p.His198Gln mutation and risk of SLE. METHODS: Forty SLE patients and 40 control subjects were recruited in this case-control study. Genotyping of C1QTNF4 p.His198Gln mutation was performed using real-time polymerase chain reaction high resolution melting method. RESULTS: We found a significant association between this mutation (GG + GC) with the risk of SLE (odds ratio = 6.33, 95% CI = 1.28-31.11). Furthermore, we observed that in the patient group, this mutation leads to early-onset SLE (19.7 ± 4.34 years for mutation carriers compared to 27.7 ± 11.4 years for wild type carriers; P = .003). CONCLUSION: Our results suggest that this mutation (p.His198Gln) potentially has an important role in SLE risk in the Iranian population.

Dynamic changes in circulating T follicular helper cell composition predict neutralising antibody responses after yellow fever vaccination.

OBJECTIVES: T follicular helper (Tfh) cells are the principal T helper cell subset that provides help to B cells for potent antibody responses against various pathogens. In this study, we took advantage of the live-attenuated yellow fever virus (YFV) vaccine strain, YF-17D, as a model system for studying human antiviral immune responses in vivo following exposure to an acute primary virus challenge under safe and highly controlled conditions, to comprehensively analyse the dynamics of circulating Tfh (cTfh) cells. METHODS: We tracked and analysed the response of cTfh and other T and B cell subsets in peripheral blood of healthy volunteers by flow cytometry over the course of 4 weeks after YF-17D vaccination. RESULTS: Using surface staining of cell activation markers to track YFV-specific T cells, we found increasing cTfh cell frequencies starting at day 3 and peaking around 2 weeks after YF-17D vaccination. This kinetic was confirmed in a subgroup of donors using MHC multimer staining for four known MHC class II epitopes of YF-17D. The subset composition of cTfh cells changed dynamically during the course of the immune response and was dominated by the cTfh1-polarised subpopulation. Importantly, frequencies of cTfh1 cells correlated with the strength of the neutralising antibody response, whereas frequencies of cTfh17 cells were inversely correlated. CONCLUSION: In summary, we describe detailed cTfh kinetics during YF-17D vaccination. Our results suggest that cTfh expansion and polarisation can serve as a prognostic marker for vaccine success. These insights may be leveraged in the future to improve current vaccine design and strategies.

HDVdb: A Comprehensive Hepatitis D Virus Database.

Hepatitis D virus (HDV) causes the most severe form of viral hepatitis, which may rapidly progress to liver cirrhosis and hepatocellular carcinoma (HCC). It has been estimated that 15-20 million people worldwide are suffering from the chronic HDV infection. Currently, no effective therapies are available to treat acute or chronic HDV infection. The remarkable sequence variability of the HDV genome, particularly within the hypervariable region has resulted in the provisional classification of eight major genotypes and various subtypes. We have developed a specialized database, HDVdb (http://hdvdb.bio.wzw.tum.de/), which contains a collection of partial and complete HDV genomic sequences obtained from the GenBank and from our own patient cohort. HDVdb enables the researchers to investigate the genetic variability of all available HDV sequences, correlation of genotypes to epidemiology and pathogenesis. Additionally, it will contribute in understanding the drug resistant mutations and develop effective vaccines against HDV infection. The database can be accessed through a web interface that allows for static and dynamic queries and offers integrated generic and specialized sequence analysis tools, such as annotation, genotyping, primer prediction, and phylogenetic analyses.

Interferon-induced protein 44-like gene promoter is differentially methylated in peripheral blood mononuclear cells of systemic lupus erythematosus patients.

BACKGROUND: The objectives of this study were to compare the interferon-induced protein 44-like (IFI44L) promoter methylation level between systemic lupus erythematosus (SLE) patients and healthy controls and to evaluate its diagnostic value in SLE. MATERIALS AND METHODS: The IFI44L promoter methylation level was measured in 49 patients with SLE and 50 healthy controls. Quantitative analysis of promoter methylation IFI44L gene in genomic DNA samples extracted from peripheral blood mononuclear cells was examined in SLE patients and healthy controls. The level of DNA methylation was compared between SLE patients and healthy controls as well as within SLE patient groups based on the presence of renal involvement. Moreover, diagnostic values of IFI44L were calculated. RESULTS: The IFI44L promoter methylation level in SLE patients was significantly lower than healthy controls (median, 43.8 vs. 57, respectively; P = 0.008). The level of IFI44L promoter methylation was not significantly different between SLE patients with renal involvement and SLE patients without renal involvement (84.6% vs. 92.7%, respectively; P = 0.774). The IFI44L promoter methylation level ≤94.3% was the best cutoff point with a sensitivity of 91.8% and a specificity of 38% to distinguish patients with SLE from healthy individuals. CONCLUSION: The level of IFI44L promoter methylation from whole peripheral blood in Iranian SLE patients was significantly lower than healthy controls. Furthermore, the DNA methylation level of IFI44L promoter was not associated with renal damage in patients with SLE.

Kinetics of hepatitis B surface antigen quasispecies during REP 2139-Ca therapy in HBeAg-positive chronic HBV infection.

Chronic HBV infection results in various clinical manifestations due to different levels of immune response. In recent years, hepatitis B treatment has improved by long-term administration of nucleos(t)ide analogues (NUCs) and peg-interferon. Nucleic acid polymers (NAPs; REP 2139-Ca and REP 2139-Mg) are new antiviral drugs that block the assembly of subviral particles, thus preventing the release of HBsAg and allowing its clearance and restoration of functional control of infection when combined with various immunotherapies. In the REP 102 study (NCT02646189), 9 of 12 patients showed substantial reduction of HBsAg and seroconversion to anti-HBs in response to REP 2139-Ca, whereas 3 of 12 patients did not show responses (>1 log reduction of HBsAg and HBV DNA from baseline). We characterized the dynamic changes of HBV quasispecies (QS) within the major hydrophilic region (MHR) of the 'pre-S/S' open reading frame including the 'a' determinant in responders and nonresponders of the REP 102 study and four untreated matched controls. HBV QS complexity at baseline varied slightly between responders and nonresponders (P = .28). However, these responders showed significant decline in viral complexity (P = .001) as REP 2139-Ca therapy progressed but no significant change in complexity was observed among the nonresponders (P = .99). The MHR mutations were more frequently observed in responders than in nonresponders and matched controls. No mutations were observed in 'a' determinant of major QS population which may interfere with the detection of HBsAg by diagnostic assays. No specific mutations were found within the MHR which could explain patients' poor HBsAg response during REP 2139-Ca therapy.

Comparison of the Effect of Disease: Modifying Antirheumatic Drugs Alone or in Combination with Biologic Drugs in the Outcome of Patients with Rheumatoid Arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a rheumatic disease that could be disabling if not treated. The aim of RA therapy is to resolve tenderness and swelling in the joints. The present study was conducted to compare two methods of RA treatment with disease-modifying anti-rheumatic drugs (DMARDs) and DMARDs with biologic drugs in two groups of patients. MATERIALS AND METHODS: The present study was a nonrandomized clinical trial which was conducted from July to September 2017 on 110 patients who were selected based on the American College of Rheumatology (2010) criteria for RA. Patients were divided into two groups of 55: Groups A and B. For the treatment of Group A, prednisolone along with one or two drugs from the DMARDs combinations was used. Group B received one biologic drug besides with the drugs of the group A. T-test and covariance analysis was used to compare the outcomes of both groups. RESULTS: Disease activity score (DAS-28) at the beginning of the study was 4.23 (0.81) in Group A and 4.51 (0.7) in Group B (P = 0.05). At the end of the study, DAS-28 was 3.52 (0.79) in Group A and 3.75 (0.85) in Group B (P = 0.1). DAS-28 activity index had a significant difference between both two groups at the beginning of the study (P = 0.05), but at the end of the study, the difference was not statistically significant (P = 0.1). CONCLUSIONS: Simultaneous use of DMARDs and biologic drugs in RA patients could lead to improvement the disease symptoms and decrease the severity and activity of the disease.

Genetic diversity of hepatitis D virus genotype-1 in Europe allows classification into subtypes.

Hepatitis delta virus (HDV) is an RNA virus which leads to both acute and chronic forms of hepatitis. At present, HDV isolates have been classified into eight major genotypes distributed over different geographical regions. Recent increase in HDV sequences in Europe and worldwide has enabled us to revisit the taxonomic classification of HDV. A total of 116 large hepatitis delta antigen (L-HDAg) nucleotide sequences and 13 full-length HDV genome sequences belonging to genotype-1 from our European cohort, as well as 621 L-HDAg nucleotide sequences belonging to genotype-1 to genotype-8 retrieved from the GenBank NCBI were included in this study. All 116 isolates of our cohort and 341 of 621 isolates (60%) account for genotype-1, while the remaining 40% of isolates were unevenly distributed across genotype-2 to genotype-8. Phylogenetic analysis of 98 L-HDAg sequences selected after elimination of redundant sequences of all 737 isolates was performed to identify plausible subtypes within HDV genotype-1. Pairwise genetic distances for L-HDAg sequences were calculated to estimate the inter-genotype and inter-subtype differences. The HDV genotype-1 isolates phylogenetically formed five distinct clusters (genotype 1a-1e), each of them corresponding to a distinct geographic region. Two distinct subtypes for HDV genotype-2 and -4 (ie -2a and -2b; -4a and -4b, respectively) could be identified based on isolate sequences from GenBank. The previously defined genotype-1 to genotype-8 have an inter-genotypic difference of ≥10%, while the newly defined subtypes of genotype-1, -2 and -4 show an inter-subtype difference of ≥3% to <10% from the average diversity. In addition, we identified unique amino acid residues, known as specificity-determining positions, amongst the proposed subtypes.

Mutations in Hepatitis D Virus Allow It to Escape Detection by CD8+ T Cells and Evolve at the Population Level.

BACKGROUND & AIMS: Hepatitis D virus (HDV) superinfection in patients with hepatitis B virus (HBV) is associated with rapid progression to liver cirrhosis and hepatocellular carcinoma. Treatment options are limited, and no vaccine is available. Although HDV-specific CD8+ T cells are thought to control the virus, little is known about which HDV epitopes are targeted by virus-specific CD8+ T cells or why these cells ultimately fail to control the infection. We aimed to define how HDV escapes the CD8+ T-cell-mediated response. METHODS: We collected plasma and DNA samples from 104 patients with chronic HDV and HBV infection at medical centers in Europe and the Middle East, sequenced HDV, typed human leukocyte antigen (HLA) class I alleles from patients, and searched for polymorphisms in HDV RNA associated with specific HLA class I alleles. We predicted epitopes in HDV that would be recognized by CD8+ T cells and corresponded with the identified virus polymorphisms in patients with resolved (n = 12) or chronic (n = 13) HDV infection. RESULTS: We identified 21 polymorphisms in HDV that were significantly associated with specific HLA class I alleles (P < .005). Five of these polymorphisms were found to correspond to epitopes in HDV that are recognized by CD8+ T cells; we confirmed that CD8+ T cells in culture targeted these HDV epitopes. HDV variant peptides were only partially cross-recognized by CD8+ T cells isolated from patients, indicating that the virus had escaped detection by these cells. These newly identified HDV epitopes were restricted by relatively infrequent HLA class I alleles, and they bound most frequently to HLA-B. In contrast, frequent HLA class I alleles were not associated with HDV sequence polymorphisms. CONCLUSIONS: We analyzed sequences of HDV RNA and HLA class I alleles that present epitope peptides to CD8+ T cells in patients with persistent HDV infection. We identified polymorphisms in the HDV proteome that associate with HLA class I alleles. Some variant peptides in epitopes from HDV were only partially recognized by CD8+ T cells isolated from patients; these could be mutations that allow HDV to escape the immune response, resulting in persistent infection. HDV escape from the immune response was associated with uncommon HLA class I alleles, indicating that HDV evolves, at the population level, to evade recognition by common HLA class I alleles.