Enhanced humoral and HLA-A2-restricted dengue virus-specific T-cell responses in humanized BLT NSG mice

Dengue is a mosquito-borne viral disease of humans, and animal models that recapitulate human immune responses or dengue pathogenesis are needed to understand the pathogenesis of the disease. We recently described an animal model for dengue virus (DENV) infection using humanized NOD-scid IL2rγ(null) mice (NSG) engrafted with cord blood haematopoietic stem cells. We sought to further improve this model by co-transplantation of human fetal thymus and liver tissues into NSG (BLT-NSG) mice. Enhanced DENV-specific antibody titres were found in the sera of BLT-NSG mice compared with human cord blood haematopoietic stem cell-engrafted NSG mice. Furthermore, B cells generated during the acute phase and in memory from splenocytes of immunized BLT-NSG mice secreted DENV-specific IgM antibodies with neutralizing activity. Human T cells in engrafted BLT-NSG mice secreted interferon-γ in response to overlapping DENV peptide pools and HLA-A2 restricted peptides. The BLT-NSG mice will allow assessment of human immune responses to DENV vaccines and the effects of previous immunity on subsequent DENV infections.     

Mucosal transmission of R5 and X4 tropic HIV-1 via vaginal and rectal routes in humanized Rag2-/- gammac -/- (RAG-hu) mice

Studies on HIV-1 mucosal transmission to evaluate early events in pathogenesis and the development of effective preventive/prophylactic methods have thus far been hampered by the lack of a suitable animal model susceptible to HIV-1 infection by either vaginal and/or rectal routes. In this regard, while primate-SIV/SHIV and cat-FIV models provided useful surrogate platforms to derive comparative data, these viruses are distinct and different from that of HIV-1. Therefore an optimal model that permits direct study of HIV-1 transmission via mucosal routes is highly desirable. The new generation of humanized NOD/SCID BLT, NOD/SCIDgammac(-/-), and Rag2(-/-)gammac(-/-) mouse models show great promise to achieve this goal. Here, we show that humanized Rag2(-/-)gammac(-/-) mice (RAG-hu) engrafted with CD34 hematopoietic progenitor cells harbor HIV-1-susceptible human cells in the rectal and vaginal mucosa and are susceptible to HIV-1 infection when exposed to cell-free HIV-1 either via vagina or rectum. Infection could be established without any prior hormonal conditioning or mucosal abrasion. Both R5 and X4 tropic viruses were capable of mucosal infection resulting in viremia and associated helper T cell depletion. There was systemic spread of the virus with infected cells detected in different organs including the intestinal mucosa. R5 virus was highly efficient in mucosal transmission by both routes whereas X4 virus was relatively less efficient in causing infection. HIV-1 infection of RAG-hu mice by vaginal and rectal routes as shown here represents the first in vivo model of HIV-1 transmission across intact mucosal barriers and as such may prove very useful for studying early events in HIV-1 pathogenesis in vivo, as well as the testing of microbicides, anti-HIV vaccines/therapeutics, and other novel strategies to prevent HIV-1 transmission.     

Humanized mice: novel model for studying mechanisms of human immune-based therapies

The lack of relevant animal models is the major bottleneck for understanding human immunology and immunopathology. In the last few years, a novel model of humanized mouse has been successfully employed to investigate some of the most critical questions in human immunology. We have set up and tested in our laboratory the latest technology for generating mice with a human immune system by reconstituting newborn immunodeficient NOD/SCID-γ ^−/−_c mice with human fetal liver-derived hematopoietic stem cells. These humanized mice have been deemed most competent as human models in a thorough comparative study with other humanized mouse technologies. Lymphocytes in these mice are of human origin while other hematopoietic cells are chimeric, partly of mouse and partly of human origin. We demonstrate that human CD8 T lymphocytes in humanized mice are fully responsive to our novel cell-based secreted heat shock protein gp96^HIV-Ig vaccine. We also show that the gp96^HIV-Ig vaccine induces powerful mucosal immune responses in the rectum and the vagina, which are thought to be required for protection from HIV infection. We posit the hypothesis that vaccine approaches tested in humanized mouse models can generate data rapidly, economically and with great flexibility (genetic manipulations are possible), to be subsequently tested in larger nonhuman primate models and humans.

     

人源化小鼠及其在病毒感染研究中的应用

人源化小鼠是指将人体细胞或组织移植入免疫缺陷小鼠,即保证不会产生免疫排斥,又可保证移植入的人体细胞/组织在小鼠体内作为合适的基质,适用于人体病原的感染及增殖,相较于普通动物模型可以获得更接近人体的较为可信的实验数据.目前人源化小鼠已经在病毒感染机制、诱导的免疫应答、抗病毒药物筛选及疫苗研发、基因治疗等方面的研究中获得应用,尤其对一些高度宿主特异性的病毒如HIV、HCV、DENV具有重大的研究价值.     

Humanized mice are susceptible to Salmonella typhi infection

Salmonella enterica serovar Typhi is a pathogen that only infects humans. Currently, there is no animal model for studying this pathogen. Recently, alymphoid RAG-2(-/-)/γ(c)(-/-) mice engrafted with human leukocytes, known as humanized mice, have been successfully utilized to develop experimental models for several human-specific viral infections, including HIV, human-like dengue fever and hepatitis C virus. Little is known about the usefulness and feasibility of the humanized mouse model for the study of human-specific bacterial pathogens, such as S. typhi. The aim of this study was to determine if Salmonella enterica serovar Typhi could establish productive infection in humanized mice. Here we report that intravenous inoculation of S. typhi into humanized mice, but not controls, established S. typhi infections. High bacterial loads were found in the liver, spleen, blood and bone marrow of mice reconstituted with human leukocytes, but not in the unreconstituted control mice. Importantly, S. typhi-infected humanized mice lost significant body weight, and some of the infected mice displayed neurological symptoms. Our data suggest, for the first time, that humanized mice are susceptible to S. typhi challenge and that this model can be utilized to study the pathogenesis of S. typhi to develop novel therapeutic strategies.     

A Hitchhiker's guide to humanized mice: new pathways to studying viral infections

Humanized mice are increasingly appreciated as an incredibly powerful platform for infectious disease research. The often very narrow species tropism of many viral infections, coupled with the sometimes misleading results from preclinical studies in animal models further emphasize the need for more predictive model systems based on human cells rather than surrogates. Humanized mice represent such a model and have been greatly enhanced with regards to their immune system reconstitution as well as immune functionality in the past years, resulting in their recommendation as a preclinical model by the US Food and Drug Administration. This review aims to give a detailed summary of the generation of human peripheral blood lymphocyte‐, CD34⁺ haematopoietic stem cell‐ and bone marrow/liver/thymus‐reconstituted mice and available improved models (e.g. myeloid‐ or T‐cell‐only mice, MISTRG, NSG‐SGM3). Additionally, we summarize human‐tropic viral infections, for which humanized mice offer a novel approach for the study of disease pathogenesis as well as future perspectives for their use in biomedical, drug and vaccine research.     

Engineering humanized mice for improved hematopoietic reconstitution

Humanized mice are immunodeficient animals engrafted with human hematopoietic stem cells that give rise to various lineages of human blood cells throughout the life of the mouse. This article reviews recent advances in the generation of humanized mice, focusing on practical considerations. We discuss features of different immunodeficient recipient mouse strains, sources of human hematopoietic stem cells, advances in expansion and genetic modification of hematopoietic stem cells, and techniques to modulate the cytokine environment of recipient mice, in order to enhance reconstitution of specific human blood lineage cells. We highlight the opportunities created by new technologies and discuss practical considerations on how to make best use of the widening array of basic models for specific research applications.     

Humanized Rag2γc (RAG-hu) mice can sustain long-term chronic HIV-1 infection lasting more than a year

HIV-1 infection is characterized by life-long viral persistence and continued decline of helper CD4 T cells. The new generation of humanized mouse models that encompass RAG-hu, hNOG and BLT mice have been shown to be susceptible to HIV-1 infection and display CD4 T cell loss. Productive infection has been demonstrated with both R5 and X4 tropic strains of HIV-1 via direct injection as well as mucosal exposure. However the duration of infection in these mice was evaluated for a limited time lasting only weeks post infection, and it is not established how long the viremia can be sustained, and if the CD4 T cell loss persists throughout the life of the infected humanized mice. In the present study we followed the HIV-1 infected RAG-hu mice to determine the long-term viral persistence and CD4 T cell levels. Our results showed that viremia persists life-long lasting for more than a year, and that CD4 T cell levels display a continuous declining trend as seen in the human. These studies provide a chronic HIV-1 infection humanized mouse model that can be used to dissect viral latency, long-term drug evaluation and immune-based therapies.     

HIV-1 immunopathogenesis in humanized mouse models

In recent years, the technology of constructing chimeric mice with humanized immune systems has markedly improved. Multiple lineages of human immune cells develop in immunodeficient mice that have been transplanted with human hematopoietic stem cells. More importantly, these mice mount functional humoral and cellular immune responses upon immunization or microbial infection. Human immunodeficiency virus type I (HIV-1) can establish an infection in humanized mice, resulting in CD4⁺ T-cell depletion and an accompanying nonspecific immune activation, which mimics the immunopathology in HIV-1-infected human patients. This makes humanized mice an optimal model for studying the mechanisms of HIV-1 immunopathogenesis and for developing novel immune-based therapies.     

Induction of human humoral immune responses in a novel HLA-DR-expressing transgenic NOD/Shi-scid/γcnull mouse

Mounting evidence has demonstrated that NOD-Shi/scid/γc(null) (NOG) mice are one of the most suitable mouse strains for humanized mouse technologies, in which various human cells or tissues can be engrafted without rejection and autonomously maintained. We have characterized and analyzed various features of the human immune system reconstituted in NOG mice by transplanting human hematopoietic stem cells (hu-HSC). One of the problems of the quasi-immune system in these hu-HSC NOG mice is that the quality of immune responses is not always sufficient, as demonstrated by the lack of IgG production in response to antigen challenge. In this study, we established a novel transgenic NOG sub-strain of mice bearing the HLA-DRA and HLA-DRB1:0405 genes, which specifically expresses HLA-DR4 molecules in MHC II-positive cells. This mouse strain enabled us to match the haplotype of HLA-DR between the recipient mice and human donor HSC. We demonstrated that T-cell homeostasis was differentially regulated in HLA-matched hu-HSC NOG mice compared with HLA-mismatched control mice, and antibody class switching was induced after immunization with exogenous antigens in HLA-matched mice. This novel mouse strain improves the reconstituted human immune systems that develop in humanized mice and will contribute to future studies of human humoral immune responses.     

登革2型病毒ZS01/01株病毒样颗粒免疫原性研究

目的 研究登革2型病毒（Dengue virus type 2,DENV-2）病毒样颗粒（virus-Like particles,VLPs）的免疫原性.方法 利用已构建的DENV-2 ZS01/01株病毒样颗粒的表达质粒转染293T细胞,对分泌型VLPs进行大量培养并通过蔗糖密度梯度离心法对其进行纯化.纯化的VLPs经Western Blot及透射电镜观察等方法鉴定后免疫BALB/c小鼠.利用ELISA及中和试验等方法对体液免疫反应进行检测,ELISPOT法测定细胞免疫水平.结果 登革2型病毒样颗粒表达质粒转染哺乳动物细胞所得上清经蔗糖密度梯度离心后,电镜下可观察到类似于天然登革病毒的大小在45～55nm之间的病毒样颗粒.体液及细胞免疫检测结果显示登革2型VLPs可以刺激小鼠产生较高水平的登革E蛋白特异性抗体及一定水平的中和抗体,免疫小鼠脾淋巴细胞经体外刺激后IFN-γ水平显著升高.结论 登革2型病毒病毒样颗粒免疫BALB/c小鼠后可引起一定水平的细胞免疫及体液免疫反应,该研究结果为四价登革病毒样颗粒疫苗的研制奠定了基础.     

Long‐term human immune system reconstitution in non‐obese diabetic (NOD)‐Rag (–)‐γ chain (–) (NRG) mice is similar but not identical to the original stem cell donor

The murine immune system is not necessarily identical to it human counterpart, which has led to the construction of humanized mice. The current study analysed whether or not a human immune system contained within the non‐obese diabetic (NOD)‐Rag1^null‐γ chain^null (NRG) mouse model was an accurate representation of the original stem cell donor and if multiple mice constructed from the same donor were similar to one another. To that end, lightly irradiated NRG mice were injected intrahepatically on day 1 of life with purified cord blood‐derived CD34⁺ stem and progenitor cells. Multiple mice were constructed from each cord blood donor. Mice were analysed quarterly for changes in the immune system, and followed for periods up to 12 months post‐transplant. Mice from the same donor were compared directly with each other as well as with the original donor. Analyses were performed for immune reconstitution, including flow cytometry, T cell receptor (TCR) and B cell receptor (BCR) spectratyping. It was observed that NRG mice could be ‘humanized’ long‐term using cord blood stem cells, and that animals constructed from the same cord blood donor were nearly identical to one another, but quite different from the original stem cell donor immune system.     

cAIMP administration in humanized mice induces a chimerization-level-dependent STING response

It is well understood that the STING signalling pathway is critical for generating a robust innate immune response to pathogens. Human and mouse STING signalling pathways are not identical, however. For example, mice lack IFI16, which has been proven important for the human STING pathway. Therefore, we investigated whether humanized mice are an appropriate experimental platform for exploring the human STING signalling cascade in vivo. We found that NOG mice reconstituted with human cord blood haematopoietic stem cells (humanized NOG mice) exhibit human STING signalling responses to an analogue of the cyclic di-nucleotide cGAMP. There was an increase in the proportions of monocytes in the lungs of mice receiving cGAMP analogue. The most robust levels of STING expression and STING-induced responses were observed in mice exhibiting the highest levels of human chimerization. Notably, differential levels of STING in lung versus spleen following cGAMP analogue treatment suggest that there are tissue-specific kinetics of STING activation and/or degradation in effector versus inductive sites. We also examined the mouse innate immune response to cGAMP analogue treatment. We detected that mouse cells in the immunodeficient NOG mice responded to the cGAMP analogue and they do so with distinct kinetics from the human response. In conclusion, humanized NOG mice represent a valuable experimental model for examining in vivo human STING responses.     

Generation of hematopoietic humanized mice in the newborn BALB/c-Rag2null Il2rγnull mouse model: a multivariable optimization approach

Hematopoietic humanized mice generated via transplantation of human hematopoietic stem cells (hHSCs) into immunodeficient mice are a valuable tool for studying development and function of the human immune system. This study was performed to generate a protocol that improves development and quality of humanized mice in the BALB/c-Rag2(null)Il2rγ(null) strain, testing route of injection, in vitro culture and freezing of hHSCs, types of cytokines in the culture, and co-injection of lineage-depleted CD34(-) cells. Specific hHSC culturing conditions and the addition of support cells were found to increase the frequency, and human hematopoietic chimerism, of humanized mice. The optimized protocol resulted in BALB/c-Rag2(null)Il2rγ(null) humanized mice displaying more consistent human hematopoietic and lymphoid engraftment. Thus, hematopoietic humanized mice generated on a BALB/c immunodeficient background represent a useful model to study the human immune system.     

Current advances in humanized mouse models

Humanized mouse models that have received human cells or tissue transplants are extremely useful in basic and applied human disease research. Highly immunodeficient mice, which do not reject xenografts and support cell and tissue differentiation and growth, are indispensable for generating additional appropriate models. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγ^null gene (e.g., NOD/SCID/γc^null and Rag2^nullγc^null mice). These strains show not only a high rate of human cell engraftment, but also generate well-differentiated multilineage human hematopoietic cells after human hematopoietic stem cell (HSC) transplantation. These humanized mice facilitate the analysis of human hematology and immunology in vivo. However, human hematopoietic cells developed from HSCs are not always phenotypically and functionally identical to those in humans. More recently, a new series of immunodeficient mice compensates for these disadvantages. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γc^null and Rag2^nullγc^null mice. In this review, we describe the current knowledge of human hematopoietic cells developed in these mice. Various human disease mouse models using these humanized mice are summarized.     

"Creation of mouse models for human diseases"

To translate research findings into medicine and drug development, we have been performing in vivo research using primary human samples. Development of mouse models with reconstituted human immunity would be critical to overcome technical and ethical constraints associated with in vivo human research. To this end, we have created humanized mice by intravenously injecting purified human hematopoietic stem cells (HSCs) into immune-compromised NOD/SCID/IL2rgKO newborns. This xenogeneic transplantation system allows long-term engraftment and multi-lineage differentiation of human HSCs. The humanized mouse fully reconstituted with human myeloid and lymphoid subsets is expected to serve as an in vivo platform for the investigation of human immune function. In addition to understanding normal human hematopoiesis and immunity, the use of humanized mice is expected to allow investigators to translate their research findings into therapeutic and pharmaceutical development. As a possibility for such translation, we developed an in vivo model of human acute myeloid leukemia (AML), a disease in which the majority of patients succumb to relapse. Using the model, we examined the pathogenesis of AML and tried to clarify the role of AML stem cells in chemotherapy resistance and relapse. Humanized mouse model for both normal and diseased immuno-hematopoietic system is opening a new era in translational medicine.     

ILC2 transfers to apolipoprotein E deficient mice reduce the lipid content of atherosclerotic lesions

The use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins’ chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known. We mutated mouse CMAH in the NOD/scid-IL2Rγc−/− (NSG) mouse strain, which is widely used for the transplantation of human cells, using the CRISPR/Cas9 system. The new strain provides a better environment for human immune cells. Transplantation of human hematopoietic stem cells leads to broad B cells repertoire, higher sensitivity to HIV-1 infection, and enhanced proliferation of transplanted peripheral blood lymphocytes. The mice showed no effect on the clearance of human immunoglobulins and enhanced transduction efficiency of recombinant adeno-associated viral vector rAAV2/DJ8. NSG-cmah−/− mice expand the mouse models suitable for human cells transplantation, and this new model has advantages in generating a human B cell repertoire. This strain is suitable to study different aspects of the human immune system development, provide advantages in patient-derived tissue and cell transplantation, and could allow studies of viral vectors and infectious agents that are sensitive to human-like sialylation of mouse glycoproteins.     

Transduction of umbilical cord blood CD34+ NOD/SCID-repopulating cells by simian foamy virus type 1 (SFV-1) vector

Foamy viruses are nonpathogenic retroviruses that offer unique opportunities for gene transfer into various cell types including hematopoietic stem cells. We used a simian foamy virus type 1 vector (SFV-1) containing a LacZ reporter gene with a titer of 1-5 x 10(6) viral particles/ml that was free of replication-competent retrovirus to transduce human umbilical cord blood CD34+ cells. Transduced CD34+ cord blood cells were transplanted into NOD/SCID mice and plated in serum-free methylcellulose culture to determine the transduction efficiency of human hematopoietic progenitor cells. A transduction efficiency of about 20% was obtained. At 6-10 weeks posttransplantation, human hematopoietic cell engraftment and marking were determined. Marrow from transplanted mice demonstrated human cell engraftment by the presence of human (CD45+) cells containing both CD19+ lymphoid and CD33+ myeloid cells. Serial sampling of NOD/SCID bone marrow revealed the presence of 6.7-14.0% CD45+ cells at 6 weeks posttransplant as compared to 3.6-27.2% CD45+ cells at 9-10 weeks posttransplant. Human progenitors examined from NOD/SCID bone marrow cells 9 weeks posttransplant revealed from 7.4 to 25.9% of the colonies exhibiting X-gal staining. Our study demonstrates the ability of a simian foamy virus vector to transduce the SCID-repopulating cell and offers a promising new gene delivery system for use in hematopoietic stem cell gene therapy.     

Humanized mouse models of HIV infection

Because of the limited tropism of HIV, in vivo modeling of this virus has been almost exclusively limited to other lentiviruses, such as simian immunodeficiency virus, that reproduce many important characteristics of HIV infection. However, there are significant genetic and biological differences among lentiviruses and some HIV-specific interventions are not effective against other lentiviruses in nonhuman hosts. For these reasons, much emphasis has recently been placed on developing alternative animal models that support HIV replication and recapitulate key aspects of HIV infection and pathogenesis in humans. Humanized mice, CD34+ hematopoietic progenitor cell transplanted immunodeficient mice, and in particular mice also implanted with human thymus/liver tissue (bone marrow liver thymus mice) that develop a functional human immune system, have been the focus of a great deal of attention as possible models to study virtually all aspects of HIV biology and pathogenesis. Humanized mice are systemically reconstituted with human lymphoid cells, offering rapid, reliable, and reproducible experimental systems for HIV research. Peripheral blood of humanized mice can be readily sampled longitudinally to assess reconstitution with human cells and to monitor HIV replication, permitting the evaluation of multiple parameters of HIV infection such as viral load levels, CD4+ T-cell depletion, immune activation, as well as the effects of therapeutic interventions. Of high relevance to HIV transmission is the extensive characterization and validation of the reconstitution with human lymphoid cells of the female reproductive tract and of the gastrointestinal tract of humanized bone marrow liver thymus mice that renders them susceptible to both vaginal and rectal HIV infection. Other important attributes of all types of humanized mice include: (i) their small size and cost that make them widely accessible; (ii) multiple cohorts of humanized mice can be made from multiple human donors and each cohort has identical human cells, permitting control of intragenetic variables; (iii) continuous de novo production of human immune cells from the transplanted CD34+ cells within each humanized mouse facilitates long-term experiments; (iv) both primary and laboratory HIV isolates can be used for experiments; and (v) in addition to therapeutic interventions, rectal and vaginal HIV prevention approaches can be studied. In summary, humanized mice can have an important role in virtually all aspects of HIV research, including the analysis of HIV replication, the evaluation of HIV restriction factors, the characterization of successful biomedical HIV prevention strategies, the evaluation of new treatment regimens, and the evaluation of novel HIV eradication strategies.     

Modulation of human beta-defensin-1 (hBD-1) in plasmacytoid dendritic cells (PDC), monocytes, and epithelial cells by influenza virus, Herpes simplex virus, and Sendai virus and its possible role in innate immunity

hBD comprise a family of antimicrobial peptides that plays a role in bridging the innate and adaptive immune responses to infection. The expression of hBD-2 increases upon stimulation of numerous cell types with LPS and proinflammatory cytokines. In contrast, hBD-1 remains constitutively expressed in most cells in spite of cytokine or LPS stimulation; however, its presence in human PDC suggests it plays a role in viral host defense. To examine this, we characterized the expression of hBD-1 in innate immune cells in response to viral challenge. PDC and monocytes increased production of hBD-1 peptide and mRNA as early as 2 h following infection of purified cells and PBMCs with PR8, HSV-1, and Sendai virus. However, treatment of primary NHBE cells with influenza resulted in a 50% decrease in hBD-1 mRNA levels, as measured by qRT-PCR at 3 h following infection. A similar inhibition occurred with HSV-1 challenge of human gingival epithelial cells. Studies with HSV-1 showed that replication occurred in epithelial cells but not in PDC. Together, these results suggest that hBD-1 may play a role in preventing viral replication in immune cells. To test this, we infected C57BL/6 WT mice and mBD-1((-/-)) mice with mouse-adapted HK18 (300 PFU/mouse). mBD-1((-/-)) mice lost weight earlier and died sooner than WT mice (P=0.0276), suggesting that BD-1 plays a role in early innate immune responses against influenza in vivo. However, lung virus titers were equal between the two mouse strains. Histopathology showed a greater inflammatory influx in the lungs of mBD-1((-/-)) mice at Day 3 postinfection compared with WT C57BL/6 mice. The results suggest that BD-1 protects mice from influenza pathogenesis with a mechanism other than inhibition of viral replication.