Higher TP53BP2 expression is associated with HBsAg loss in peginterferon-α-treated patients with chronic hepatitis B.

BACKGROUND & AIMS: HBsAg loss is only observed in a small proportion of patients with chronic hepatitis B (CHB) who undergo interferon treatment. Investigating the host factors crucial for functional cure of CHB can aid in identifying individuals who would benefit from peginterferon-α (Peg-IFNα) therapy. METHODS: We conducted a genome-wide association study (GWAS) by enrolling 48 patients with CHB who achieved HBsAg loss after Peg-IFNα treatment and 47 patients who didn't. In the validation stage, we included 224 patients, of whom 90 had achieved HBsAg loss, to validate the identified significant single nucleotide polymorphisms. To verify the functional involvement of the candidate genes identified, we performed a series of in vitro and in vivo experiments. RESULTS: GWAS results indicated a significant association between the rs7519753 C allele and serum HBsAg loss in patients with CHB after Peg-IFNα treatment (p = 4.85 × 10-8, odds ratio = 14.47). This association was also observed in two independent validation cohorts. Expression quantitative trait locus analysis revealed higher hepatic TP53BP2 expression in individuals carrying the rs7519753 C allele (p = 2.90 × 10-6). RNA-sequencing of liver biopsies from patients with CHB after Peg-IFNα treatment revealed that hepatic TP53BP2 levels were significantly higher in the HBsAg loss group compared to the HBsAg persistence group (p = 0.035). In vitro and in vivo experiments demonstrated that loss of TP53BP2 decreased interferon-stimulated gene levels and the anti-HBV effect of IFN-α. Mechanistically, TP53BP2 was found to downregulate SOCS2, thereby facilitating JAK/STAT signaling. CONCLUSION: The rs7519753 C allele is associated with elevated hepatic TP53BP2 expression and an increased probability of serum HBsAg loss post-Peg-IFNα treatment in patients with CHB. TP53BP2 enhances the response of the hepatocyte to IFN-α by suppressing SOCS2 expression. IMPACT AND IMPLICATIONS: Chronic hepatitis B (CHB) remains a global public health issue. Although current antiviral therapies are more effective in halting disease progression, only a few patients achieve functional cure for hepatitis B with HBsAg loss, highlighting the urgent need for a cure for CHB. This study revealed that the rs7519753 C allele, which is associated with high expression of hepatic TP53BP2, significantly increases the likelihood of serum HBsAg loss in patients with CHB undergoing Peg-IFNα treatment. This finding not only provides a promising predictor for HBsAg loss but identifies a potential therapeutic target for Peg-IFNα treatment. We believe our results are of great interest to a wide range of stakeholders based on their potential clinical implications.

A novel hepatitis B virus capsid assembly modulator QL-007 inhibits HBV replication and infection through altering capsid assembly.

The core protein allosteric modulators (CpAMs) have shown great potential as highly effective antiviral drugs against hepatitis B virus (HBV) in preclinical studies and clinical trials. In this study, we evaluated a small molecule compound called QL-007, which could potentially influence capsid assembly, using HBV replicated and susceptible cell models as well as mice infected with rAAV-HBV. QL-007 significantly inhibited HBV replication in a dose-dependent manner both in vitro and in vivo, resulting in significant decreases in HBV DNA, 3.5 kb HBV RNA and HBeAg. Furthermore, QL-007 not only induced the formation of misshaped Cp149 capsids but also possessed the capability to disassemble HBV capsids. It is noteworthy that QL-007 effectively reduced cccDNA biosynthesis in de novo infections. Mechanistically, QL-007 blocked the encapsidation of pgRNA and induced aberrant polymers assembly at concentrations ≥100 nM, while having no impact on the stability of core proteins. In conclusion, our findings underscore the potential of QL-007 as an effective agent against HBV replication and introduce it as a novel CpAM for the antiviral treatment of chronic hepatitis B.

Personalized neoantigen pulsed dendritic cell vaccine for advanced lung cancer.

Neoantigens are considered to be ultimate target of tumor immunotherapy due to their high tumor specificity and immunogenicity. Dendritic cell (DCs) vaccines based on neoantigens have exciting effects in treatment of some malignant tumors and are a promising therapeutic modality. Lung cancer is a lethal disease with the highest morbidity and mortality rate in the world. Despite the rapid development of targeted therapy and immune checkpoint inhibitors for lung cancer in recent years, their efficacy is still unsatisfactory overall. Therefore, there is an urgent unmet clinical need for lung cancer treatment. Here, we attempted to treat lung cancer using a personalized neoantigen peptide-pulsed autologous DC vaccine and conducted a single-arm, 2 medical centers, pilot study initiated by the investigator (ChiCTR-ONC-16009100, NCT02956551). The patients enrolled were patients with heavily treated metastatic lung cancer. Candidate neoantigens were derived from whole-exome sequencing and RNA sequencing of fresh biopsy tissues as well as bioinformatics analysis. A total of 12 patients were enrolled in this study. A total of 85 vaccine treatments were administered with a median value of 5 doses/person (range: 3-14 doses/person). In total, 12-30 peptide-based neoantigens were selected for each patient. All treatment-related adverse events were grade 1-2 and there were no delays in dosing due to toxic effects. The objective effectiveness rate was 25%; the disease control rate was 75%; the median progression-free survival was 5.5 months and the median overall survival was 7.9 months. This study provides new evidence for neoantigen vaccine therapy and new therapeutic opportunities for lung cancer treatment.

The Unique, the Known, and the Unknown of Spumaretrovirus Assembly.

Within the family of Retroviridae, foamy viruses (FVs) are unique and unconventional with respect to many aspects in their molecular biology, including assembly and release of enveloped viral particles. Both components of the minimal assembly and release machinery, Gag and Env, display significant differences in their molecular structures and functions compared to the other retroviruses. This led to the placement of FVs into a separate subfamily, the Spumaretrovirinae. Here, we describe the molecular differences in FV Gag and Env, as well as Pol, which is translated as a separate protein and not in an orthoretroviral manner as a Gag-Pol fusion protein. This feature further complicates FV assembly since a specialized Pol encapsidation strategy via a tripartite Gag-genome-Pol complex is used. We try to relate the different features and specific interaction patterns of the FV Gag, Pol, and Env proteins in order to develop a comprehensive and dynamic picture of particle assembly and release, but also other features that are indirectly affected. Since FVs are at the root of the retrovirus tree, we aim at dissecting the unique/specialized features from those shared among the Spuma- and Orthoretrovirinae. Such analyses may shed light on the evolution and characteristics of virus envelopment since related viruses within the Ortervirales, for instance LTR retrotransposons, are characterized by different levels of envelopment, thus affecting the capacity for intercellular transmission.

TruNeo: an integrated pipeline improves personalized true tumor neoantigen identification.

BACKGROUND: Neoantigen-based personal vaccines and adoptive T cell immunotherapy have shown high efficacy as a cancer treatment in clinical trials. Algorithms for the accurate prediction of neoantigens have played a pivotal role in such studies. Some existing bioinformatics methods, such as MHCflurry and NetMHCpan, identify neoantigens mainly through the prediction of peptide-MHC binding affinity. However, the predictive accuracy of immunogenicity of these methods has been shown to be low. Thus, a ranking algorithm to select highly immunogenic neoantigens of patients is needed urgently in research and clinical practice. RESULTS: We develop TruNeo, an integrated computational pipeline to identify and select highly immunogenic neoantigens based on multiple biological processes. The performance of TruNeo and other algorithms were compared based on data from published literature as well as raw data from a lung cancer patient. Recall rate of immunogenic ones among the top 10-ranked neoantigens were compared based on the published combined data set. Recall rate of TruNeo was 52.63%, which was 2.5 times higher than that predicted by MHCflurry (21.05%), and 2 times higher than NetMHCpan 4 (26.32%). Furthermore, the positive rate of top 10-ranked neoantigens for the lung cancer patient were compared, showing a 50% positive rate identified by TruNeo, which was 2.5 times higher than that predicted by MHCflurry (20%). CONCLUSIONS: TruNeo, which considers multiple biological processes rather than peptide-MHC binding affinity prediction only, provides prioritization of candidate neoantigens with high immunogenicity for neoantigen-targeting personalized immunotherapies.

The chromatin binding domain, including the QPQRYG motif, of feline foamy virus Gag is required for viral DNA integration and nuclear accumulation of Gag and the viral genome.

The retroviral Gag protein, the major component of released particles, plays different roles in particle assembly, maturation or infection of new host cells. Here, we characterize the Gag chromatin binding site including the highly conserved QPQRYG motif of feline foamy virus, a member of the Spumaretrovirinae. Mutagenesis of critical residues in the chromatin binding site/QPQRYG motif almost completely abrogates viral DNA integration and reduces nuclear accumulation of Gag and viral DNA. Genome packaging, reverse transcription, particle release and uptake into new target cells are not affected. The integrity of the QPQRYG motif appears to be important for processes after cytosolic entry, likely influencing incoming virus capsids or disassembly intermediates but not Gag synthesized de novo in progeny virus-producing cells. According to our data, chromatin binding is a shared feature among foamy viruses but further work is needed to understand the mechanisms involved.

Replacement of feline foamy virus bet by feline immunodeficiency virus vif yields replicative virus with novel vaccine candidate potential.

BACKGROUND: Hosts are able to restrict viral replication to contain virus spread before adaptive immunity is fully initiated. Many viruses have acquired genes directly counteracting intrinsic restriction mechanisms. This phenomenon has led to a co-evolutionary signature for both the virus and host which often provides a barrier against interspecies transmission events. Through different mechanisms of action, but with similar consequences, spumaviral feline foamy virus (FFV) Bet and lentiviral feline immunodeficiency virus (FIV) Vif counteract feline APOBEC3 (feA3) restriction factors that lead to hypermutation and degradation of retroviral DNA genomes. Here we examine the capacity of vif to substitute for bet function in a chimeric FFV to assess the transferability of anti-feA3 factors to allow viral replication. RESULTS: We show that vif can replace bet to yield replication-competent chimeric foamy viruses. An in vitro selection screen revealed that an engineered Bet-Vif fusion protein yields suboptimal protection against feA3. After multiple passages through feA3-expressing cells, however, variants with optimized replication competence emerged. In these variants, Vif was expressed independently from an N-terminal Bet moiety and was stably maintained. Experimental infection of immunocompetent domestic cats with one of the functional chimeras resulted in seroconversion against the FFV backbone and the heterologous FIV Vif protein, but virus could not be detected unambiguously by PCR. Inoculation with chimeric virus followed by wild-type FFV revealed that repeated administration of FVs allowed superinfections with enhanced antiviral antibody production and detection of low level viral genomes, indicating that chimeric virus did not induce protective immunity against wild-type FFV. CONCLUSIONS: Unrelated viral antagonists of feA3 cellular restriction factors can be exchanged in FFV, resulting in replication competence in vitro that was attenuated in vivo. Bet therefore may have additional functions other than A3 antagonism that are essential for successful in vivo replication. Immune reactivity was mounted against the heterologous Vif protein. We conclude that Vif-expressing FV vaccine vectors may be an attractive tool to prevent or modulate lentivirus infections with the potential option to induce immunity against additional lentivirus antigens.

Mutagenesis of N-terminal residues of feline foamy virus Gag reveals entirely distinct functions during capsid formation, particle assembly, Gag processing and budding.

BACKGROUND: Foamy viruses (FVs) of the Spumaretrovirinae subfamily are distinct retroviruses, with many features of their molecular biology and replication strategy clearly different from those of the Orthoretroviruses, such as human immunodeficiency, murine leukemia, and human T cell lymphotropic viruses. The FV Gag N-terminal region is responsible for capsid formation and particle budding via interaction with Env. However, the critical residues or motifs in this region and their functional interaction are currently ill-defined, especially in non-primate FVs. RESULTS: Mutagenesis of N-terminal Gag residues of feline FV (FFV) reveals key residues essential for either capsid assembly and/or viral budding via interaction with the FFV Env leader protein (Elp). In an in vitro Gag-Elp interaction screen, Gag mutations abolishing particle assembly also interfered with Elp binding, indicating that Gag assembly is a prerequisite for this highly specific interaction. Gradient sedimentation analyses of cytosolic proteins indicate that wild-type Gag is mostly assembled into virus capsids. Moreover, proteolytic processing of Gag correlates with capsid assembly and is mostly, if not completely, independent from particle budding. In addition, Gag processing correlates with the presence of packaging-competent FFV genomic RNA suggesting that Pol encapsidation via genomic RNA is a prerequisite for Gag processing. Though an appended heterogeneous myristoylation signal rescues Gag particle budding of mutants unable to form capsids or defective in interacting with Elp, it fails to generate infectious particles that co-package Pol, as evidenced by a lack of Gag processing. CONCLUSIONS: Changes in proteolytic Gag processing, intracellular capsid assembly, particle budding and infectivity of defined N-terminal Gag mutants highlight their essential, distinct and only partially overlapping roles during viral assembly and budding. Discussion of these findings will be based on a recent model developed for Gag-Elp interactions in prototype FV.

Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy.

The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.

Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple.

Proanthocyanidins (PAs) are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis.

An apple MYB transcription factor, MdMYB3, is involved in regulation of anthocyanin biosynthesis and flower development.

BACKGROUND: Red coloration of fruit is an important trait in apple, and it is mainly attributed to the accumulation of anthocyanins, a class of plant flavonoid metabolites. Anthocyanin biosynthesis is genetically determined by structural and regulatory genes. Plant tissue pigmentation patterns are mainly controlled by expression profiles of regulatory genes. Among these regulatory genes are MYB transcription factors (TFs), wherein the class of two-repeats (R2R3) is deemed the largest, and these are associated with the anthocyanin biosynthesis pathway. Although three MdMYB genes, almost identical in nucleotide sequences, have been identified in apple, it is likely that there are other R2R3 MYB TFs that are present in the apple genome that are also involved in the regulation of coloration of red color pigmentation of the skin of apple fruits. RESULTS: In this study, a novel R2R3 MYB gene has been isolated and characterized in apple. This MYB gene is closely related to the Arabidopsis thaliana AtMYB3, and has been designated as MdMYB3. This TF belongs to the subgroup 4 R2R3 family of plant MYB transcription factors. This apple MdMYB3 gene is mapped onto linkage group 15 of the integrated apple genetic map. Transcripts of MdMYB3 are detected in all analyzed tissues including leaves, flowers, and fruits. However, transcripts of MdMYB3 are higher in excocarp of red-skinned apple cultivars than that in yellowish-green skinned apple cultivars. When this gene is ectopically expressed in Nicotiana tabacum cv. Petite Havana SR1, flowers of transgenic tobacco lines carrying MdMYB3 have exhibited increased pigmentation and accumulate higher levels of anthocyanins and flavonols than wild-type flowers. Overexpression of MdMYB3 has resulted in transcriptional activation of several flavonoid pathway genes, including CHS, CHI, UFGT, and FLS. Moreover, peduncles of flowers and styles of pistils of transgenic plants overexpressing MdMYB3 are longer than those of wild-type plants, thus suggesting that this TF is involved in regulation of flower development. CONCLUSIONS: This study has identified a novel MYB transcription factor in the apple genome. This TF, designated as MdMYB3, is involved in transcriptional activation of several flavonoid pathway genes. Moreover, this TF not only regulates the accumulation of anthocyanin in the skin of apple fruits, but it is also involved in the regulation of flower development, particularly that of pistil development.

[Anti-HBV effect of nucleotide analogues on mouse model of chronic HBV infection mediated by recombinant adeno-associated virus 8].

We evaluated the anti-HBV effects of nucleotide analogues, Entecavir (ETV) and Lamivudine (LAM) targeting mouse model of HBV persistent infection with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV). Ninety percent (27 of 30 mice) of rAAVS-treated mice were chosen as mouse model. Four groups were orally administrated with different doses of ETV (1 mg/(kgd) or 0.1 mg/(kgd)) and LAM (500 mg/(kgd) or 100 mg/(kgd)) once a day for 10 days. The other two groups were set as normal saline treated and untreated control. We detected the levels of HBV DNA, HBeAg and HBsAg in sera at different time. Results indicate that HBV DNA level decreased significantly (P < 0.05) in drug-treated groups compared with normal saline group after drug administration. Fifteen days after the drug withdrawal, HBV DNA level rebounded back obviously (P < 0.05) in groups with low doses of ETV and LAM. However, there was no apparent change of HBeAg and HBsAg in the whole process among all groups. These results showed that our model could reflect the anti-viral effect of nucleotide analogues. This model can be a useful and convenient tool for anti-HBV drug discovery.

Prevalence of Helicobacter pylori vacA, cagA and iceA genotypes and correlation with clinical outcome.

The aim of this study was to assess the genetic status of cagA, vacA subtype and iceA genotypes of Helicobacter pylori and the relationship with upper gastrointestinal diseases in Northeast China. Gastric biopsies were obtained from 378 patients with upper gastrointestinal diseases and 197 samples were used. The cagA, vacA alleles and iceA genotypes were determined by polymerase chain reaction. CagA was present in 176 (89.3%) of 197 patients. Of the 197 cases, 186 (94.4%) had vacA signal sequence s1c allele, 6 (3%) had s1a and 5 (2.5%) had s1b. The vacA s2 genotype was not detected in our study. VacA middle region sequences, m1 and m2, were found in 20 (10.2%) and 150 (76.1%), respectively. The allelic variant iceA1 (70.1%) was more prevalent than iceA2 (23.4%). The vacA allele s1am2 had a significant relationship with the presence of gastric cancer (p<0.05) and the iceA1 genotype was also associated with gastric cancer (p<0.05). These may be useful risk factors for upper gastrointestinal diseases.

[Study on the differences of two mouse models of hepatitis B virus infection by transduction with rAAV8-1. 3HBV].

We recently developed a mouse model of hepatitis B virus (HBV) chronic infection by intravenous (i.v.) injection with rAAV8-1. 3HBV to C57BL/6 mice. To define the responses of different mouse strains after injection with rAAV8-1. 3HBV, we intravenously injected rAAV8-1. 3HBV at doses of 4 x10(9) (Viral genome,vg), 4 x 10(10) vg and 4 x 10(11) vg to C57BL/6 and BALB/c mice,respectively, and determined the levels of serum HBV antigen and antibody by ELISA,serum viral DNA by real-time PCR,and HBcAg expression in liver by immunohistochemical staining. For C57BL/6 mouse strain with injection of rAAV8-1. 3HBV at three doses, 100% of the mice carried HBV for more than 8 months. The levels of serum HBsAg and HBeAg, serum viral DNA and HBcAg-positive hepatocytes increased in a rAAV8-1. 3HBV dose-dependent manner. For C57BL/6 mice injected with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg,over 40% of hepatocytes expressed HBcAg and serum viral DNA reached over 10(5) IU/mL. No HBV antibody was detected in sera of C57BL/6 mice. For BALB/c mice with injection of rAAV8-1. 3HBV at three doses, serum HBeAg, serum viral DNA and HBcAg-positive hepatocytes persisted for more than 8 months, but serum HBsAg declined remarkably at 2 weeks after injection. The levels of serum HBeAg and HBcAg-positive hepatocytes in BALB/c mice increased in a rAAV8-1. 3HBV dose-dependent manner. Injection with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg resulted in over 50% of BALB/c mice hepatocytes expressing HBcAg. Serum anti-HBsAg were detected in BALB/c mice with rAAV8-1. 3HBV injection at the dose of 4 x10 (10) vg. In conclusion, both C57BL/6 and BALB/c strains can be developed to chronic HBV infection mouse models by i. v. injection with rAAV8-1. 3HBV at doses of 4 x10(9) - 4 x 10(11) vg and the levels of HBV replication increase in a rAAV8-1. 3HBV dose-dependent manner. In contrast to C57BL/6 strain, the BALB/c mice carry out humoral immunity to HBsAg, but fail to mediate HBV clearance.