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The chemotherapy of retinoblastoma (RB), a malignant ocular childhood disease, is often limited by the development of resistance against commonly used drugs. We identified inositol polyphosphate 4-phosphatase type II (INPP4B) as a differentially regulated gene in etoposide-resistant RB cell lines, potentially involved in the development of RB resistances. INPP4B is controversially discussed as a tumor suppressor and an oncogenic driver in various cancers, but its role in retinoblastoma in general and chemoresistant RB in particular is yet unknown. In the study presented, we investigated the expression of INPP4B in RB cell lines and patients and analyzed the effect of INPP4B overexpression on etoposide resistant RB cell growth in vitro and in vivo. INPP4B mRNA levels were significantly downregulated in RB cells lines compared to the healthy human retina, with even lower expression levels in etoposide-resistant compared to the sensitive cell lines. Besides, a significant increase in INPP4B expression was observed in chemotherapy-treated RB tumor patient samples compared to untreated tumors. INPP4B overexpression in etoposide-resistant RB cells resulted in a significant reduction in cell viability with reduced growth, proliferation, anchorage-independent growth, and in ovo tumor formation. Caspase-3/7-mediated apoptosis was concomitantly increased, suggesting a tumor suppressive role of INPP4B in chemoresistant RB cells. No changes in AKT signaling were discernible, but p-SGK3 levels increased following INPP4B overexpression, indicating a potential regulation of SGK3 signaling in etoposide-resistant RB cells. RNAseq analysis of INPP4B overexpressing, etoposide-resistant RB cell lines revealed differentially regulated genes involved in cancer progression, mirroring observed in vitro and in vivo effects of INPP4B overexpression and strengthening INPP4B’s importance for cell growth control and tumorigenicity.

Neonatal and infant immune responses are characterized by a limited capability to generate protective Ab titers and memory B cells as seen in adults. Multiple studies support an immature or even impaired character of umbilical cord blood (UCB) B cells themselves. In this study, we provide a comprehensive molecular and functional comparison of B cell subsets from UCB and adult peripheral blood. Most UCB B cells have a mature, naive B cell phenotype as seen in adults. The UCB Ig repertoire is highly variable but interindividually conserved, as BCR clonotypes are frequently shared between neonates. Furthermore, UCB B cells show a distinct transcriptional program that confers accelerated responsiveness to stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into Ab-secreting cells, presumably limiting memory B cell formation. Humanized mice suggest that the distinctness of UCB versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice, albeit our findings suggest only partial comparability to murine B-1 cells. Our study shows that UCB B cells are not immature or impaired but differ from their adult mature counterpart in a conserved BCR repertoire, efficient IgA class switching, and accelerated, likely transient response dynamics.

Background: Critically ill patients, especially those who have undergone solid organ transplantation (SOT), are at risk of invasive pulmonary aspergillosis (IPA). The outcome relevance of adequately treated putative IPA (pIPA) is a matter of debate. The aim of this study is to assess the outcome relevance of pIPA in a cohort of critically ill patients with and without SOT. Methods: Data from 121 surgical critically ill patients with pIPA (n = 30) or non-pIPA (n = 91) were included. Cox regression analysis was used to identify risk factors for mortality and unfavourable outcomes after 28 and 90 days. Results: Mortality rates at 28 days were similar across the whole cohort of patients (pIPA: 31% vs. non-pIPA: 27%) and did not differ in the subgroup of patients after SOT (pIPA: 17% vs. non-pIPA: 22%). A higher Sequential Organ Failure Assessment (SOFA) score and evidence of bacteraemia were identified as risk factors for mortality and unfavourable outcome, whereas pIPA itself was not identified as an independent predictor for poor outcomes. Conclusions: Adequately treated pIPA did not increase the risk of death or an unfavourable outcome in this mixed cohort of critically ill patients with or without SOT, whereas higher disease severity and bacteraemia negatively affected the outcome. OA Förderung 2023

Sepsis is associated with profound immune dysregulation that increases the risk for life-threatening secondary infections: Dendritic cells (DCs) undergo functional reprogramming due to yet unknown changes during differentiation in the bone marrow (BM). In parallel, lymphopenia and exhaustion of T lymphocytes interfere with antigen-specific adaptive immunity. We hypothesized that there exists a link between T cells and the modulation of DC differentiation in the BM during murine polymicrobial sepsis. Sepsis was induced by cecal ligation and puncture (CLP), a model for human bacterial sepsis. At different time points after CLP, the BM and spleen were analyzed in terms of T-cell subpopulations, activation, and Interferon (IFN)-γ synthesis as well as the number of pre-DCs. BM-derived DCs were generated

Single-cell RNAseq/VDJseq of tumor cells and normal residual B (NRB) cells from peripheral blood of chronic lymphocytic leukemia (CLL) patients identified three distinct tumor subsets according to phenotype, transcriptome, and immunoglobulin-V-gene (IgV)-mutations. Two major subsets share a typical CLL phenotype but differ in signaling, metabolism and cell cycle control, indicating that the circulating CLL pool is shaped by two states of activity. The third CLL subset shows the phenotype, proliferation capacity and extensive IgV-mutation diversity of normal CD5+ memory B cells. This previously unrecognized CLL tumor subset, which intermingles with NRB cells, was confirmed in 33 IgV-mutated (M-CLL) and IgV-unmutated (U-CLL) cases. Longitudinal IgV-mutation phylogenetics suggest that these NRB-associated CLL cells are generated pathogenetically early, mostly in germinal center reactions, and archive the individual IgV-diversification program, which is conserved throughout CLL course. Our study suggests that diversity is established early in CLL, that each tumor is composed of multiple subclonal expansions, and subclonal evolution can be depicted by IgV-mutation phylogenetics.

Kibler et al. show that circulating memory B cells are systematically and comprehensively archived in the human spleen throughout life. The random, dynamic priming of archive members determines the composition of human splenic marginal zone B cells., Human memory B cells (MBCs) are generated and diversified in secondary lymphoid tissues throughout the organism. A paired immunoglobulin (Ig)-gene repertoire analysis of peripheral blood (PB) and splenic MBCs from infant, adult, and elderly humans revealed that throughout life, circulating MBCs are comprehensively archived in the spleen. Archive MBC clones are systematically preserved and uncoupled from class-switching. Clonality in the spleen increases steadily, but boosts at midlife, thereby outcompeting small clones. The splenic marginal zone (sMZ) represents a primed MBC compartment, generated from a stochastic exchange within the archive memory pool. This is supported by functional assays, showing that PB and splenic CD21+ MBCs acquire transient CD21high expression upon NOTCH2-stimulation. Our study provides insight that the human MBC system in PB and spleen is composed of three interwoven compartments: the dynamic relationship of circulating, archive, and its subset of primed (sMZ) memory changes with age, thereby contributing to immune aging., Graphical Abstract

Objective: Developing an integrative approach to early treatment response classification using survival modeling and bioinformatics with various biomarkers for early assessment of filgrastim (granulocyte colony stimulating factor) treatment effects in amyotrophic lateral sclerosis (ALS) patients. Filgrastim, a hematopoietic growth factor with excellent safety, routinely applied in oncology and stem cell mobilization, had shown preliminary efficacy in ALS. Methods: We conducted individualized long-term filgrastim treatment in 36 ALS patients. The PRO-ACT database, with outcome data from 23 international clinical ALS trials, served as historical control and mathematical reference for survival modeling. Imaging data as well as cytokine and cellular data from stem cell analysis were processed as biomarkers in a non-linear principal component analysis (NLPCA) to identify individual response. Results: Cox proportional hazard and matched-pair analyses revealed a significant survival benefit for filgrastim-treated patients over PRO-ACT comparators. We generated a model for survival estimation based on patients in the PRO-ACT database and then applied the model to filgrastim-treated patients. Model-identified filgrastim responders displayed less functional decline and impressively longer survival than non-responders. Multimodal biomarkers were then analyzed by PCA in the context of model-defined treatment response, allowing identification of subsequent treatment response as early as within 3 months of therapy. Strong treatment response with a median survival of 3.8 years after start of therapy was associated with younger age, increased hematopoietic stem cell mobilization, less aggressive inflammatory cytokine plasma profiles, and preserved pattern of fractional anisotropy as determined by magnetic resonance diffusion tensor imaging (DTI-MRI). Conclusion: Long-term filgrastim is safe, is well-tolerated, and has significant positive effects on disease progression and survival in a small cohort of ALS patients. Developing and applying a model-based biomarker response classification allows use of multimodal biomarker patterns in full potential. This can identify strong individual treatment responders (here: filgrastim) at a very early stage of therapy and may pave the way to an effective individualized treatment option.

The human infant B cell system is considered premature or impaired. Here we show that most cord blood B cells are mature and functional as seen in adults, albeit with distinct transcriptional programs providing accelerated responsiveness to T cell-independent and T cell-dependent stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into antibody-secreting cells, thereby presumably limiting memory B cell formation. The neonatal Ig-repertoire is highly variable, but conserved, showing recurrent B cell receptor (BCR) clonotypes frequently shared between neonates. Our study demonstrates that cord blood B cells are not impaired but differ from their adult counterpart in a conserved BCR repertoire and rapid but transient response dynamics. These properties may account for the sensitivity of neonates to infections and limited effectivity of vaccination strategies. Humanized mice suggest that the distinctness of cord blood versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice. Still, our findings reveal overall limited comparability of human cord blood B cells and murine B-1 cells.Significance StatementNeonates and infants suffer from enhanced susceptibility to infections. Our study contrasts with the current concept of a premature or impaired B cell system in neonates, by showing that most cord blood B cells are mature and functional. However, their responses are rapid but provide only short-term protection, which may help to improve infant vaccination strategies. We propose an altered perspective on the early human B cell system, which looks similar to but functions differently from the adult counterpart. Finally, our analysis indicates that cord blood- and adult B cell development occur layered as in mice, but certain mouse models still may offer a limited view on human neonatal B cell immunity.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by age-inappropriate symptoms of inattention and/or hyperactivity and impulsivity. ADHD is highly prevalent in childhood and often persists into adulthood. Both genetic variants and environmental factors play a role in the onset and persistence of ADHD, and epigenetic changes, such as DNA methylation are considered as a link for their interplay. To investigate this, we studied DNA methylation in 37 candidate genes by performing targeted bisulfite sequencing of DNA isolated from whole blood of N = 88 individuals diagnosed with adult ADHD and N = 91 unaffected individuals (mean age 34.2 years). Differentially methylated sites were assessed by generalized linear models testing ADHD status and ADHD symptoms, accounting for a methylation-based smoking score, age, sex, and blood cell count. DNA methylation of single sites within DRD4 and KLDR1 was associated with adult ADHD status, and multiple DNA methylation sites within TARBP1 were associated with ADHD symptoms in adulthood and childhood. Awaiting replication, findings of this pilot study point to TARBP1 as a new candidate gene for ADHD symptoms. Our work also stresses the need for research to further examine the effects of environmental factors, such as nicotine exposure, on epigenetic modifications associated with psychiatric traits.

Genetic factors may be involved in the onset of neurodegenerative diseases like Alzheimer's disease. In the case of the familial type, the disease is due to an inherited mutation at specific sites in three genes. Also, there are some genetic risk factors that facilitate the development of sporadic Alzheimer's disease. All of these genetic analyses were performed using blood samples as a source of DNA. However, the presence of somatic mutations in the brain can be identified only using brain samples. In this review, we comment on a method that correctly identifies single nucleotide variations in the human brain and that can be used to validate high-through sequencing techniques. This method involves selective enrichment of the DNA population bearing the nucleotide variations, thereby facilitating posterior validation of the data by Sanger's sequencing.

Background & Aims\ud\udHepatitis 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.\ud\ud\udMethods\ud\udWe 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.\ud\ud\udResults\ud\udWe 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.\ud\ud\udConclusions\ud\udWe 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.

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.

Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection. IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.

Antiretroviral treatment of Human Immunodeficiency Virus type-1 (HIV-1) infections with CCR5-antagonists requires the co-receptor usage prediction of viral strains. Currently available tools are mostly designed based on subtype B strains and thus are in general not applicable to non-B subtypes. However, HIV-1 infections caused by subtype B only account for approximately 11% of infections worldwide. We evaluated the performance of several sequence-based algorithms for co-receptor usage prediction employed on subtype A V3 sequences including circulating recombinant forms (CRFs) and subtype C strains. We further analysed sequence profiles of gp120 regions of subtype A, B and C to explore functional relationships to entry phenotypes. Our analyses clearly demonstrate that state-of-the-art algorithms are not useful for predicting co-receptor tropism of subtype A and its CRFs. Sequence profile analysis of gp120 revealed molecular variability in subtype A viruses. Especially, the V2 loop region could be associated with co-receptor tropism, which might indicate a unique pattern that determines co-receptor tropism in subtype A strains compared to subtype B and C strains. Thus, our study demonstrates that there is a need for the development of novel algorithms facilitating tropism prediction of HIV-1 subtype A to improve effective antiretroviral treatment in patients.

OA gold Antiretroviral treatment of Human Immunodeficiency Virus type-1 (HIV-1) infections with CCR5-antagonists requires the co-receptor usage prediction of viral strains. Currently available tools are mostly designed based on subtype B strains and thus are in general not applicable to non-B subtypes. However, HIV-1 infections caused by subtype B only account for approximately 11% of infections worldwide. We evaluated the performance of several sequence-based algorithms for co-receptor usage prediction employed on subtype A V3 sequences including circulating recombinant forms (CRFs) and subtype C strains. We further analysed sequence profiles of gp120 regions of subtype A, B and C to explore functional relationships to entry phenotypes. Our analyses clearly demonstrate that state-of-the-art algorithms are not useful for predicting co-receptor tropism of subtype A and its CRFs. Sequence profile analysis of gp120 revealed molecular variability in subtype A viruses. Especially, the V2 loop region could be associated with co-receptor tropism, which might indicate a unique pattern that determines co-receptor tropism in subtype A strains compared to subtype B and C strains. Thus, our study demonstrates that there is a need for the development of novel algorithms facilitating tropism prediction of HIV-1 subtype A to improve effective antiretroviral treatment in patients.

OA Förderung 2015 High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches (“AmpliconDuo filter”). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.

Our knowledge about the clonal composition and intraclonal diversity of the human memory B-cell compartment and the relationship between memory B-cell subsets is still limited, although these are central issues for our understanding of adaptive immunity. We performed a deep sequencing analysis of rearranged immunoglobulin (Ig) heavy chain genes from biological replicates, covering more than 100,000 memory B lymphocytes from two healthy adults. We reveal a highly similar B-cell receptor repertoire among the four main human IgM(+) and IgG(+) memory B-cell subsets. Strikingly, in both donors, 45% of sequences could be assigned to expanded clones, demonstrating that the human memory B-cell compartment is characterized by many, often very large, B-cell clones. Twenty percent of the clones consisted of class switched and IgM(+)(IgD(+)) members, a feature that correlated significantly with clone size. Hence, we provide strong evidence that the vast majority of Ig mutated B cells--including IgM(+)IgD(+)CD27(+) B cells--are post-germinal center (GC) memory B cells. Clone members showed high intraclonal sequence diversity and high intraclonal versatility in Ig class and IgG subclass composition, with particular patterns of memory B-cell clone generation in GC reactions. In conclusion, GC produce amazingly large, complex, and diverse memory B-cell clones, equipping the human immune system with a versatile and highly diverse compartment of IgM(+)(IgD(+)) and class-switched memory B cells.

HIV-1 evolves strongly and undergoes geographic differentiation as it spreads in diverse host populations around the world. For instance, distinct genomic backgrounds can be observed between the pandemic subtype B, prevalent in Europe and North-America, and its offspring clade B’ in East Asia. Here we ask whether this differentiation affects the selection pressure experienced by the virus. To answer this question we evaluate selection pressure on the HIV-1 envelope protein gp120 at the level of individual codons using a simple and fast estimation method based on the ratio k a /k s of amino acid changes to synonymous changes. To validate the approach we compare results to those from a state-of-the-art mixed-effect method. The agreement is acceptable, but the analysis also demonstrates some limitations of the simpler approach. Further, we find similar distributions of codons under stabilizing and directional selection pressure in gp120 for subtypes B and B’ with more directional selection pressure in variable loops and more stabilizing selection in the constant regions. Focusing on codons with increased k a /k s values in B’, we show that these codons are scattered over the whole of gp120, with remarkable clusters of higher density in regions flanking the variable loops. We identify a significant statistical association of glycosylation sites and codons with increased k a /k s values.

Poster-Abstract

Long term antiviral therapy with nucleoside/nucleotide analogs have been routinely used to treat chronic hepatitis B virus (HBV) infection but may lead to the emergence of drug-resistant viral mutants. However, the HBV resistance mutations for tenofovir (TDF) remain controversial. It is speculated that the genetic barrier for TDF resistance may be high for HBV. We asked whether selected amino acid substitutions in HBV polymerase may reduce susceptibility to TDF. A series of amino acids in HBV polymerase were selected based on bioinformatics analysis for mutagenesis. The replication competence and susceptibility to TDF of the mutated HBV clones were determined both in vitro and in vivo. nineteen mutations in HBV polymerase were included and impaired the replication competence of HBV genome in different degrees. The mutations at rtL77F (sS69C), rtF88L (sF80Y), and rtP177G (sR169G) also significantly affected HBsAg expression. The HBV mutants with rtP177G and rtF249A were found to have reduced susceptibility to TDF in vitro with a resistance index of 2.53 and 12.16, respectively. The testing in in vivo model based on the hydrodynamic injection revealed the antiviral effect of TDF against wild type and mutated HBV genomes and confirmed the reduced the susceptibility of mutant HBV to TDF.