Combining serum microRNAs and machine learning algorithms for diagnosing infectious fever after HSCT.

Infection post-hematopoietic stem cell transplantation (HSCT) is one of the main causes of patient mortality. Fever is the most crucial clinical symptom indicating infection. However, current microbial detection methods are limited. Therefore, timely diagnosis of infectious fever and administration of antimicrobial drugs can effectively reduce patient mortality. In this study, serum samples were collected from 181 patients with HSCT with or without infection, as well as the clinical information. And more than 80 infectious-related microRNAs in the serum were selected according to the bulk RNA-seq result and detected in the 345 time-pointed serum samples by Q-PCR. Unsupervised clustering result indicates a close association between these microRNAs expression and infection occurrence. Compared to the uninfected cohort, more than 10 serum microRNAs were identified as the combined diagnostic markers in one formula constructed by the Random Forest (RF) algorithms, with a diagnostic accuracy more than 0.90. Furthermore, correlations of serum microRNAs to immune cells, inflammatory factors, pathgens, infection tissue, and prognosis were analyzed in the infection cohort. Overall, this study demonstrates that the combination of serum microRNAs detection and machine learning algorithms holds promising potential in diagnosing infectious fever after HSCT.

Functions and Clinical Relevance of Liver-Derived Immunoglobulins.

Liver is the largest internal organ of the body with vital functions. In addition to its endocrine and exocrine activities, liver also plays a pivotal role in the immune system, including haematopoietic functions. Liver parenchymal cells, which are epithelial cells, have been found to possess innate immune functions by expressing pattern-recognition receptors (PRRs), producing complement components, and secreting cytokines. Intriguingly, in recent years, it has been discovered that liver epithelial cells also produce immunoglobulins (Igs), which have long been thought to be produced exclusively by B cells. Notably, even liver epithelial cells from B lymphocyte-deficient mice, including SCID mice and µMT mice, could also produce Igs. Compelling evidence has revealed both the physiological and pathological functions of liver-derived Igs. For instance, liver epithelial cells-derived IgM can serve as a source of natural and specific antibodies that contribute to innate immune responses, while liver-produced IgG can act as a growth factor to promote cell proliferation and survival in normal hepatocytes and hepatocarcinoma. Similar to that in B cells, the toll-like receptor 9 (TLR9)-MyD88 signaling pathway is also actively involved in promoting liver epithelial cells to secrete IgM. Liver-derived Igs could potentially serve as biomarkers, prognostic indicators, and therapeutic targets in the clinical setting, particularly for liver cancers and liver injury. Nevertheless, despite significant advances, much remains unknown about the mechanisms governing Ig transcription in liver cells, as well as the detailed functions of liver-derived Igs and their involvement in diseases and adaptive immunity. Further studies are still needed to reveal these underlying, undefined issues related to the role of liver-derived Igs in both immunity and diseases.

Comparison of Non B-Ig and B-Ig.

Immunoglobulin (Ig) has been widely acknowledged to be produced solely by B-lineage cells. However, growing evidence has demonstrated the expression of Ig in an array of cancer cells, as well as normal cells including epithelial cells, epidermal cells, mesangial cells, monocytes, and neutrophils. Ig has even been found to be expressed in non-B cells at immune-privileged sites such as neurons and spermatogenic cells. Despite these non-B cell-derived Igs (non-B-Igs) sharing the same symmetric structures with conventional Igs (B-Igs), further studies have revealed unique characteristics of non-B-Ig, such as restricted variable region and aberrant glycosylation. Moreover, non-B-Ig exhibits properties of promoting malignant behaviours of cancer cells, therefore it could be utilised in the clinic as a potential therapeutic biomarker or target. The elucidation of the generation and regulation of non-B-Ig will certainly broaden our understanding of immunology.

Non B Cell-Derived Immunoglobulins in Lung Epithelial Cells and Lung Cancer.

As the locus for air exchange, lung tissue is perpetually exposed to a significant quantity of foreign pathogens. Consequently, lung has developed a refined and intricate immune system. Beyond their physical and chemical barrier roles, lung epithelial cells can contribute to immune defence through the expression of Toll-like receptors (TLRs) and other pattern recognition receptors, along with the secretion of cytokines. Emerging evidence demonstrates that lung epithelial cells can generate and secrete immunoglobulins (Igs), including IgM, IgA, or IgG, thus performing antibody function. Moreover, malignantly transformed lung epithelial cells have been discovered to produce high levels of Ig, predominantly IgG, which do not fulfill the role of antibodies, but instead carries out tumour-promoting activity. Structural analysis has indicated that the biological activity of IgG produced by lung cancer cells differs from that of Igs produced by normal lung epithelial cells due to the unique glycosylation modification. Specifically, the sialylated IgG (SIA-IgG), characterised by a non-traditional N-glycosylation modification at the Asn162 site of Igγ CH1, is highly expressed in tumour stem cells. It has been demonstrated that SIA-IgG relies on this unique sialylation modification to promote tumorigenesis, metastasis, and immune evasion. Current results have proven that the Ig produced by lung epithelial cells has multifaceted biological activities, including immune defence functions under physiological conditions, while acquiring tumour-promoting activity during malignant transformation. These insights possess potential for the diagnosis and treatment of lung cancer as novel biomarkers and targets.

[Research progress on the relationship between neural synchronization activity and memory consolidation during non-rapid eye movement sleep].

Neural synchronization activity is considered a key aspect of information processing in the nervous system. Local synchronization within different frequency ranges and inter-regional synchronization are ubiquitous and related to various behavioral and cognitive functions. As memory is a higher cognitive function of the brain, the formation and consolidation of memory are closely related to neural synchronization activity. This article provides an overview of the research progress on the relationship between neural synchronization activity and memory consolidation, focusing primarily on the neuro-oscillatory activities across multiple brain regions during non-rapid eye movement (NREM) sleep in vivo, as well as the synchronous burst activity in cultured neural networks in vitro. Finally, we analyzed the existing issues in current research and provided a perspective on future relevant studies.

Inhibition of antigen presentation during AAV gene therapy using virus peptides.

The clinical trial using adeno-associated virus (AAV) vector delivery of mini-dystrophin in patients with Duchenne Muscular Dystrophy (DMD) demonstrated a cytotoxic lymphocyte (CTL) response targeting the transgene product. These mini-dystrophin-specific T-cells have the potential to clear all transduced muscle, presenting the general gene therapy concern of overcoming the CTL response to foreign proteins that provide therapeutic benefit. In this study, we exploited a natural immunosuppression strategy employed by some viruses that results in CTL evasion only in transduced cells. After transfection of the plasmids encoding viral peptides and ovalbumin, which includes the immune-domain epitope SIINFEKL, several viral small peptides (ICP47 and US6) inhibited the SIINFEKL peptide presentation. A single AAV vector genome that consisted of either transgene AAT fused with SIINFEKL epitope and, separately, ICP47 expressed from different promoters or a single fusion protein with ICP47 linked by a furin cleavage peptide (AATOVA-ICP47) decreased antigen presentation. Compared with AAV/AATOVA in which decreased AAT expression was observed at late time points, persistent transgene expression was obtained after systemic administration of AAV/AATOVA-ICP47 vectors in mice. We extended this strategy to DMD gene therapy. After administration of AAV vector encoding human mini-dystrophin fusion protein with ICP47 into mdx mice, a lower mini-dystrophin-specific CTL response was induced. Importantly, the ICP47 fusion to mini-dystrophin inhibited CTLs mediated cytotoxicity. Although demonstrated herein using AAT and mini-dystrophin transgenes in an AAV context, the collective results have implications for all gene therapy applications resulting in foreign peptides by immune suppression in only genetically modified cells.

Hepatocyte-Derived Igκ Exerts a Protective Effect against ConA-Induced Acute Liver Injury.

Immunoglobulin (Igκ) has been reported to be expressed in sorted liver epithelial cells of µMT mice, and the sequence characteristics of hepatocyte-derived Igκ were different from those of classical B-cell-derived Igκ. However, the physiological function of hepatocyte-derived Igκ is still unclear. The expression of Igκ was firstly identified in primary hepatocytes and normal liver cell line (NCTC1469), and hepatocyte-derived Igκ expression was elevated and displayed unique localization in hepatocytes of concanavalin A (ConA)-induced hepatitis model. Moreover, Igκ knockout mice were more sensitive to ConA-induced hepatitis and had higher serum aspartate aminotransferase (AST) levels, more severe histological injury and a greater number of terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive cells as compared with littermate controls. Furthermore, knockdown of Igκ in primary hepatocytes and NCTC1469 cells led to accelerated activation of the mitochondrial death pathway and caspase-3 cleavage in vitro, which might be related to inhibition of NF-κB signaling pathway and activation of JNK via the cytoskeleton dynamics. Taken together, these results indicate that hepatocyte-derived Igκ mediates cellular resistance to ConA-induced liver injury by inhibiting activation of caspase-3 and the mitochondrial death pathway, suggesting that Igκ plays an important role in hepatocyte survival and exerts a protective effect against ConA-induced liver injury in mice.

Superior human hepatocyte transduction with adeno-associated virus vector serotype 7.

Although therapeutic outcomes have been achieved in hemophilia patients after delivery of clotting factor genes to the liver using adeno-associated virus (AAV) vectors, it is well known that the preclinical results generated from hemophilia animal models have not been directly predictive of successful translation in humans. To address this discrepancy humanized mouse models have recently been used to predict AAV transduction efficiency for human hepatocytes. In this study we evaluated AAV vector transduction from several serotypes in human liver hepatocytes xenografted into chimeric mice. After systemic administration of AAV vectors encoding a GFP transgene in humanized mice, the liver was harvested for either immunohistochemistry staining or flow cytometry assay for AAV human hepatocyte transduction analysis. We observed that AAV7 consistently transduced human hepatocytes more efficiently than other serotypes in both immunohistochemistry assay and flow cytometry analysis. To better assess the future application of AAV7 for systemic administration in the treatment of hemophilia or other liver diseases, we analyzed the prevalence of neutralizing antibodies (NAbs) to AAV7 in sera from healthy subjects and patients with hemophilia. In the general population, the prevalence of NAbs to AAV7 was lower than that of AAV2 or AAV3B. However, a higher prevalence of AAV7 NAbs was found in patients with hemophilia. In summary, results from this study suggest that AAV7 vectors should be considered as an effective vehicle for human liver targeting in future clinical trials.

Binding of the monoclonal antibody RP215 to immunoglobulin G in metastatic lung adenocarcinomas is correlated with poor prognosis.

AIMS: Cancer cell-derived immunoglobulin (Ig)G (cancer-IgG) has been found to be involved in the pathogenesis and progression of many cancers, including lung cancer. The aim of the present study was to investigate the relationship between cancer-IgG expression in lung adenocarcinoma (ADC) and clinicopathological characteristics and clinical outcome. METHODS AND RESULTS: Immunohistochemical analysis was performed using an RP215 monoclonal antibody to determine cancer-IgG expression in 140 lung ADC patients. Cell migration and invasion were analysed in A549 cell line after short interfering RNA (siRNA) knockdown of IgG and cell sorting by flow cytometry. Our results show that RP215 immunostaining score is correlated significantly with local invasion (P < 0.05) and tumour differentiation (P < 0.05) in ADC. Moreover, RP215 staining was significantly higher in metastatic tumours than in primary tumours (P < 0.0001). The knockdown of IgG resulted in a reduction of cell migration and invasion. In contrast, RP215-positive cells displayed greater migration and invasion ability than RP215-negative cells. Additionally, a higher RP215 immunostaining score was associated significantly with poor prognosis. CONCLUSIONS: RP215 staining is correlated strongly with differentiation, local invasion, metastasis and clinical outcome of patients with lung ADC. Our results suggest that RP215 can serve as a biomarker for prognosis of lung ADC.

IgG and IgA with potential microbial-binding activity are expressed by normal human skin epidermal cells.

The innate immune system of the skin is thought to depend largely on a multi-layered mechanical barrier supplemented by epidermis-derived antimicrobial peptides. To date, there are no reports of antimicrobial antibody secretion by the epidermis. In this study, we report the expression of functional immunoglobulin G (IgG) and immunoglobulin A (IgA), previously thought to be only produced by B cells, in normal human epidermal cells and the human keratinocyte line HaCaT. While B cells express a fully diverse Ig, epidermal cell-expressed IgG or IgA showed one or two conservative VHDJH rearrangements in each individual. These unique VDJ rearrangements in epidermal cells were found neither in the B cell-derived Ig VDJ databases published by others nor in our positive controls. IgG and IgA from epidermal cells of the same individual had different VDJ rearrangement patterns. IgG was found primarily in prickle cells, and IgA was mainly detected in basal cells. Both epidermal cell-derived IgG and IgA showed potential antibody activity by binding pathogens like Staphylococcus aureus, the most common pathogenic skin bacteria, but the microbial-binding profile was different. Our data indicates that normal human epidermal cells spontaneously express IgG and IgA, and we speculate that these Igs participate in skin innate immunity.

Hypoxia and hypoxia-inducible factor-1α provoke toll-like receptor signalling-induced inflammation in rheumatoid arthritis.

OBJECTIVES: Hyperplasia of synovial fibroblasts, infiltration with lymphocytes and tissue hypoxia are major characteristics of rheumatoid arthritis (RA). Extensive data support a key role for toll-like receptors (TLRs) in RA. Little is known regarding the impact of hypoxia on TLR-induced inflammation in RA. The aim of this study was to reveal the effects of hypoxia and its regulator, hypoxia-inducible factor-1α (HIF-1α), on the inflammatory response of RA synovial fibroblasts (RASF) to TLR ligands. METHODS: Hypoxia was induced in RASF by incubation with Na2S2O4. TLR3 ligand polyIC, TLR2 ligand peptidoglycan, TLR4 ligand LPS and TLR9 ligand CpG were used to stimulate the cells. Effects of hypoxia on TLR-induced inflammatory mediators were determined by RT-PCR, qPCR and ELISA. Overexpression of HIF-1α as well as knocking-down its expression was used to reveal its fundamental role. RASF-induced inflammatory T cell expansion was determined by flow cytometry analysis of T helper (Th)1/Th17 cells, and IFN-γ/IL-17 production by ELISA after RASF/T cell coculture. RESULTS: Hypoxia potentiated the expression of inflammatory cytokines, metalloproteinases and VEGF in RASF stimulated by different TLR ligands, especially polyIC, a synthetic mimic of dsRNA from viruses or apoptotic cells. HIF-1α played a fundamental role in this synergy. Moreover, HIF-1α overexpression enhanced RASF-mediated expansion of inflammatory Th1 and Th17 cells, leading to proinflammatory IFN-γ and IL-17 production. CONCLUSIONS: Our findings suggest that hypoxia and HIF-1α may function in conjunction with TLR-stimulated innate immune responses to drive inflammation in RA. This pathway may serve as a therapeutic target for the disease.

Evidence of IgY subclass diversification in snakes: evolutionary implications.

Mammalian IgG and IgE are thought to have evolved from IgY of nonmammalian tetrapods; however, no diversification of IgY subclasses has been reported in reptiles or birds, which are phylogenetically close to mammals. To our knowledge, we report the first evidence of the presence of multiple IgY-encoding (υ) genes in snakes. Two υ genes were identified in the snake Elaphe taeniura, and three υ genes were identified in the Burmese python (Python molurus bivittatus). Although four of the υ genes displayed a conventional four-H chain C region exon structure, one of the υ genes in the Burmese python lacked the H chain C region 2 exon, thus exhibiting a structure similar to that of the mammalian γ genes. We developed mouse mAbs specific for the IgY1 and IgY2 of E. taeniura and showed that both were expressed in serum; each had two isoforms: one full-length and one truncated at the C terminus. The truncation was not caused by alternative splicing or transcriptional termination. We also identified the µ and δ genes, but no α gene, in both snakes. This study provides valuable clues for our understanding of Ig gene evolution in tetrapods.

Decoding the connection between SLE and DNA Sensors: A comprehensive review.

Systemic lupus erythematosus (SLE) is recognized as a prevalent autoimmune disorder characterized by a multifaceted pathogenesis potentially influenced by a combination of environmental factors, genetic predisposition, and hormonal regulation. The continuous study of immune system activation is especially intriguing. Analysis of blood samples from individuals with SLE reveals an abnormal increase in interferon levels, along with the existence of anti-double-stranded DNA antibodies. This evidence suggests that the development of SLE may be initiated by innate immunity. The presence of abnormal dsDNA fragments can activate DNA sensors within cells, particularly immune cells, leading to the initiation of downstream signaling cascades that result in the upregulation of relevant cytokines and the subsequent initiation of adaptive immune responses, such as B cell differentiation and T cell activation. The intricate pathogenesis of SLE results in DNA sensors exhibiting a wide range of functions in innate immune responses that are subject to variation based on cell types, developmental processes, downstream effector signaling pathways and other factors. The review aims to reorganize how DNA sensors influence signaling pathways and contribute to the development of SLE according to current studies, with the aspiration of furnishing valuable insights for future investigations into the pathological mechanisms of SLE and potential treatment approaches.

An investigation of the immune epitopes of adeno-associated virus capsid-derived peptides among hemophilia patients.

Adeno-associated virus (AAV) is an optimal gene vector for monogenic disorders. However, neutralizing antibodies (Nabs) against AAV hinder its widespread application in gene therapy. In this study, we biosynthesized peptides recognized by the binding antibodies (Babs) from the sera containing high Nab titers against AAV2. We established four immunological methods to detect immune epitopes of the AAV2-derived peptides, including a Bab assay, Nab assay, B cell receptor (BCR) detecting assay, and immunoglobin-producing B cell enzyme-linked immunosorbent spot (B cell ELISpot) assay. Correlations among the epitopes determined by these four methods were analyzed using the serum samples and peripheral blood mononuclear cells (PBMC) from 89 patients with hemophilia A/B. As decoys, the peptides' ability to block the Nab of AAV2 particles was assessed using AAV transduction models both in vitro and in vivo. Overall, we provide insights into AAV2-capsid-derived peptide immune epitopes, involving the Nab, Bab, BCR, and B cell ELISpot assays, offering alternative immunological evaluation approaches and strategies to overcome Nab barriers in AAV-mediated gene therapy.

Innate Immune Response to Viral Vectors in Gene Therapy.

Viral vectors play a pivotal role in the field of gene therapy, with several related drugs having already gained clinical approval from the EMA and FDA. However, numerous viral gene therapy vectors are currently undergoing pre-clinical research or participating in clinical trials. Despite advancements, the innate response remains a significant barrier impeding the clinical development of viral gene therapy. The innate immune response to viral gene therapy vectors and transgenes is still an important reason hindering its clinical development. Extensive studies have demonstrated that different DNA and RNA sensors can detect adenoviruses, adeno-associated viruses, and lentiviruses, thereby activating various innate immune pathways such as Toll-like receptor (TLR), cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING), and retinoic acid-inducible gene I-mitochondrial antiviral signaling protein (RLR-MAVS). This review focuses on elucidating the mechanisms underlying the innate immune response induced by three widely utilized viral vectors: adenovirus, adeno-associated virus, and lentivirus, as well as the strategies employed to circumvent innate immunity.

Human Hepatocyte Transduction with Adeno-Associated Virus Vector.

As the adeno-associated virus (AAV) vectors hold unique advantages over other viral vectors, AAV gene therapy has accumulated rapid progress and development. Liver-targeted gene therapy by AAV vectors has been successfully applied in clinical trials for many diseases. Low transduction efficiency and high prevalence of neutralizing antibodies (Nabs), however, are the major obstacles to further translate this therapeutic strategy into clinical trials. Pre-clinical evaluation on hepatocytes could help to elucidate the tropism of AAV serotypes for liver-targeted gene therapy, and could also provide a test model to develop novel AAV mutants with Nabs evasion and high liver tropism. Here, we described the basic laboratory procedure to apply the AAV vector to transduce human hepatocytes in vitro and in vivo with some tips gained from our own experience.

Chimeric Mice Engrafted With Canine Hepatocytes Exhibits Similar AAV Transduction Efficiency to Hemophilia B Dog.

Adeno-associated virus (AAV) mediated gene therapy has been successfully applied in clinical trials, including hemophilia. Novel AAV vectors have been developed with enhanced transduction and specific tissue tropism. Considering the difference in efficacy of AAV transduction between animal models and patients, the chimeric xenograft mouse model with human hepatocytes has unique advantages of studying AAV transduction efficiency in human hepatocytes. However, it is unclear whether the results in humanized mice can predict AAV transduction efficiency in human hepatocytes. To address this issue, we studied the AAV transduction efficacy in canine hepatocytes in both canine hepatocyte xenografted mice and real dogs. After administration of AAV vectors from different serotypes into canine hepatocyte xenograft mice, AAV8 induced the best canine hepatocyte transduction followed by AAV9, then AAV3, 7, 5 and 2. After administration of AAV/cFIX (cFIX-opt-R338L) vectors in hemophilia B dogs, consistent with the result in chimeric mice, AAV8 induced the highest cFIX protein expression and function, followed by AAV9 and then AAV2. These results suggest that mice xenografted with hepatocytes from different species could be used to predict the AAV liver transduction in real species and highlight this potential platform to explore novel AAV variants for future clinical applications.

Learning populations with hubs govern the initiation and propagation of spontaneous bursts in neuronal networks after learning.

Spontaneous bursts in neuronal networks with propagation involving a large number of synchronously firing neurons are considered to be a crucial feature of these networks both in vivo and in vitro. Recently, learning has been shown to improve the association and synchronization of spontaneous events in neuronal networks by promoting the firing of spontaneous bursts. However, little is known about the relationship between the learning phase and spontaneous bursts. By combining high-resolution measurement with a 4,096-channel complementary metal-oxide-semiconductor (CMOS) microelectrode array (MEA) and graph theory, we studied how the learning phase influenced the initiation of spontaneous bursts in cultured networks of rat cortical neurons in vitro. We found that a small number of selected populations carried most of the stimulus information and contributed to learning. Moreover, several new burst propagation patterns appeared in spontaneous firing after learning. Importantly, these "learning populations" had more hubs in the functional network that governed the initiation of spontaneous burst activity. These results suggest that changes in the functional structure of learning populations may be the key mechanism underlying increased bursts after learning. Our findings could increase understanding of the important role that synaptic plasticity plays in the regulation of spontaneous activity.

NBIGV-DB: A dedicated database of non-B cell derived immunoglobulin variable region.

Immunoglobulins (Ig) are important immune molecules that possess highly diverse variable region sequences enabling antigen recognition. According to classical immune theory, B lymphocytes have been considered the only source of Ig production (B-Igs). However, accumulating evidence have suggested that Igs are also produced by many non-B cells (non-B Igs), including epithelial cells, neurons, germ cells, as well as myeloid cells of hemopoietic system. Besides acting as bona fide antibodies, Non-B Igs have alternative cellular functions, such as promotion of cell survival, adhesion and migration. More importantly, Unlike the unlimited sequence diversity of B-Igs, the non-B Igs exhibit conserved V(D)J patterns across the same lineages. To support the analysis and comparison of variable region sequences from Igs, produced by B and non-B cells, we established a database (NBIGV) constituted by a non-B Ig variable region repertoire, which includes 727,989 VHDJH and VκJκ recombination sequences of non-B Igs sequenced from mouse samples. Upon database search, users can view, browse and investigate the variable region sequences of non-B Igs according to respective mice strains and tissues as well as Ig classes. Moreover, users can easily download selected sequences and/or compare sequences of interest with known non-B Ig sequences present in the database using NCBI-BLAST algorithms. Additionally, our database integrates a submission page and supplementary sample information. The NBIGV database may serve as a valuable resource for sequence analyses of Non-B Igs. NBIGV database is freely available at http://nbigv.org.

Development of AAV Variants with Human Hepatocyte Tropism and Neutralizing Antibody Escape Capacity.

Adeno-associated virus (AAV) vectors have been successfully used in patients with bleeding disorders and blindness. For human liver targeting, two major factors restrict effective AAV transduction after systemic administration of AAV vectors: human hepatocyte tropism and neutralizing antibodies (Nabs). In this study, we attempted to isolate AAV variants with the ability to transduce human hepatocytes and escape Nabs using a directed evolution approach in vivo. After four cycles of selection, 14 AAV capsid mutants were identified from a capsid shuffling library selected in the presence of human Intravenous Immunoglobulin (IVIG) and isolated from human hepatocytes xenografted into chimeric mice. AAV neutralization assays using IVIG showed that most of the mutants showed the Nab escape pattern in a manner similar to that of AAV8 or AAV9 and better than that of other AAV serotypes. Different mutants displayed varying capacities to escape Nab activity from individual serum samples collected from healthy subjects or hemophilia patients. The mutant AAV LP2-10 was found in 12 colonies out of 25, which was composed of capsids from AAV serotypes 2, 6, 8, and 9, with VP3 subunits derived from AAV8 swapped with AAV6 from residues 261 to 272. The mutant AAV LP2-10 manifested a higher ability than that of other serotypes to escape Nabs in IVIG and most human serum samples. After injection of AAV vectors encoding a self-complementary GFP cassette into chimeric mice, LP2-10 transduced human hepatocytes with efficiency similar to that of AAV8. In summary, AAV mutants can be isolated in humanized mice with both human hepatocyte tropism and the ability to evade Nab activity.