Modeling the Tumor Microenvironment and Cancer Immunotherapy in Next-Generation Humanized Mice.

Cancer immunotherapy has brought significant clinical benefits to numerous patients with malignant disease. However, only a fraction of patients experiences complete and durable responses to currently available immunotherapies. This highlights the need for more effective immunotherapies, combination treatments and predictive biomarkers. The molecular properties of a tumor, intratumor heterogeneity and the tumor immune microenvironment decisively shape tumor evolution, metastasis and therapy resistance and are therefore key targets for precision cancer medicine. Humanized mice that support the engraftment of patient-derived tumors and recapitulate the human tumor immune microenvironment of patients represent a promising preclinical model to address fundamental questions in precision immuno-oncology and cancer immunotherapy. In this review, we provide an overview of next-generation humanized mouse models suitable for the establishment and study of patient-derived tumors. Furthermore, we discuss the opportunities and challenges of modeling the tumor immune microenvironment and testing a variety of immunotherapeutic approaches using human immune system mouse models.

SARS-CoV-2 Variant-Specific Infectivity and Immune Profiles Are Detectable in a Humanized Lung Mouse Model.

Small animal models that accurately model pathogenesis of SARS-CoV-2 variants are required for ongoing research efforts. We modified our human immune system mouse model to support replication of SARS-CoV-2 by implantation of human lung tissue into the mice to create TKO-BLT-Lung (L) mice and compared infection with two different variants in a humanized lung model. Infection of TKO-BLT-L mice with SARS-CoV-2 recapitulated the higher infectivity of the B.1.1.7 variant with more animals becoming infected and higher sustained viral loads compared to mice challenged with an early B lineage (614D) virus. Viral lesions were observed in lung organoids but no differences were detected between the viral variants as expected. Partially overlapping but distinct immune profiles were also observed between the variants with a greater Th1 profile in VIDO-01 and greater Th2 profile in B.1.1.7 infection. Overall, the TKO-BLT-L mouse supported SARS-CoV-2 infection, recapitulated key known similarities and differences in infectivity and pathogenesis as well as revealing previously unreported differences in immune responses between the two viral variants. Thus, the TKO-BLT-L model may serve as a useful animal model to study the immunopathobiology of newly emerging variants in the context of genuine human lung tissue and immune cells.

Discovery and characterization of SARS-CoV-2 reactive and neutralizing antibodies from humanized CAMouseHG mice through rapid hybridoma screening and high-throughput single-cell V(D)J sequencing.

The coronavirus disease 2019 pandemic has caused more than 532 million infections and 6.3 million deaths to date. The reactive and neutralizing fully human antibodies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are effective detection tools and therapeutic measures. During SARS-CoV-2 infection, a large number of SARS-CoV-2 reactive and neutralizing antibodies will be produced. Most SARS-CoV-2 reactive and neutralizing fully human antibodies are isolated from human and frequently encoded by convergent heavy-chain variable genes. However, SARS-CoV-2 viruses can mutate rapidly during replication and the resistant variants of neutralizing antibodies easily survive and evade the immune response, especially in the face of such focused antibody responses in humans. Therefore, additional tools are needed to develop different kinds of fully human antibodies to compensate for current deficiency. In this study, we utilized antibody humanized CAMouseHG mice to develop a rapid antibody discovery method and examine the antibody repertoire of SARS-CoV-2 RBD-reactive hybridoma cells derived from CAMouseHG mice by using high-throughput single-cell V(D)J sequencing analysis. CAMouseHG mice were immunized by 28-day rapid immunization method. After electrofusion and semi-solid medium screening on day 12 post-electrofusion, 171 hybridoma clones were generated based on the results of SARS-CoV-2 RBD binding activity assay. A rather obvious preferential usage of IGHV6-1 family was found in these hybridoma clones derived from CAMouseHG mice, which was significantly different from the antibodies found in patients with COVID-19. After further virus neutralization screening and antibody competition assays, we generated a noncompeting two-antibody cocktail, which showed a potent prophylactic protective efficacy against SARS-CoV-2 in cynomolgus macaques. These results indicate that humanized CAMouseHG mice not only provide a valuable platform to obtain fully human reactive and neutralizing antibodies but also have a different antibody repertoire from humans. Thus, humanized CAMouseHG mice can be used as a good complementary tool in discovery of fully human therapeutic and diagnostic antibodies.

Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2.HLA-DR4.Rag1KO.IL-2RγcKO.NOD) for COVID-19.

We report a Human Immune System (HIS)-humanized mouse model ("DRAGA": HLA-A2.HLA-DR4.Rag1KO.IL-2 RγcKO.NOD) for COVID-19 research. DRAGA mice express transgenically HLA-class I and class-II molecules in the mouse thymus to promote human T cell development and human B cell Ig-class switching. When infused with human hematopoietic stem cells from cord blood reconstitute a functional human immune system, as well as human epi/endothelial cells in lung and upper respiratory airways expressing the human ACE2 receptor for SARS-CoV-2. The DRAGA mice were able to sustain SARS-CoV-2 infection for at least 25 days. Infected mice showed replicating virus in the lungs, deteriorating clinical condition, and human-like lung immunopathology including human lymphocyte infiltrates, microthrombi and pulmonary sequelae. Among the intra-alveolar and peri-bronchiolar lymphocyte infiltrates, human lung-resident (CD103+) CD8+ and CD4+ T cells were sequestered in epithelial (CD326+) lung niches and secreted granzyme B and perforin, suggesting anti-viral cytotoxic activity. Infected mice also mounted human IgG antibody responses to SARS-CoV-2 viral proteins. Hence, HIS-DRAGA mice showed unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathological mechanisms and testing the safety and efficacy of candidate vaccines and therapeutics.

Humanized mouse models for preclinical evaluation of HIV cure strategies.

Although the world is currently focused on the COVID-19 pandemic, HIV/AIDS remains a significant threat to public health. To date, the HIV/AIDS pandemic has claimed the lives of over 36 million people, while nearly 38 million people are currently living with the virus. Despite the undeniable success of antiretroviral therapy (ART) in controlling HIV, the medications are not curative. Soon after initial infection, HIV integrates into the genome of infected cells as a provirus, primarily, within CD4+ T lymphocytes and tissue macrophages. When not actively transcribed, the provirus is referred to as a latent reservoir because it is hidden to the immune system and ART. Following ART discontinuation, HIV may emerge from the replication-competent proviruses and resumes the infection of healthy cells. Thus, these latent reservoirs are a major obstacle to an HIV cure, and their removal remains a priority. A vital aspect in the development of curative therapies is the demonstration of efficacy in an animal model, such as the humanized mouse model. Therefore, optimization, standardization, and validation of the humanized mouse model are a priority. The purpose of this review article is to provide an update on existing humanized mouse models, highlighting the advantages and disadvantages of each as they pertain to HIV cure studies and to review the approaches to curative therapies that are under investigation.

Humanized mice reveal a macrophage-enriched gene signature defining human lung tissue protection during SARS-CoV-2 infection.

The human immunological mechanisms defining the clinical outcome of SARS-CoV-2 infection remain elusive. This knowledge gap is mostly driven by the lack of appropriate experimental platforms recapitulating human immune responses in a controlled human lung environment. Here, we report a mouse model (i.e., HNFL mice) co-engrafted with human fetal lung xenografts (fLX) and a myeloid-enhanced human immune system to identify cellular and molecular correlates of lung protection during SARS-CoV-2 infection. Unlike mice solely engrafted with human fLX, HNFL mice are protected against infection, severe inflammation, and histopathological phenotypes. Lung tissue protection from infection and severe histopathology associates with macrophage infiltration and differentiation and the upregulation of a macrophage-enriched signature composed of 11 specific genes mainly associated with the type I interferon signaling pathway. Our work highlights the HNFL model as a transformative platform to investigate, in controlled experimental settings, human myeloid immune mechanisms governing lung tissue protection during SARS-CoV-2 infection.

The mouse resource at National Resource Center for Mutant Mice.

Mouse models are essential for dissecting disease mechanisms and defining potential drug targets. There are more than 18,500 mouse strains available for research communities in National Resource Center for Mutant Mice (NRCMM) of China, affiliated with Model Animal Research Center of Nanjing University and Gempharmatech Company. In 2019, Gempharmatech launched the Knockout All Project (KOAP) aiming to generate null mutants and gene floxed strains for all protein-coding genes in mouse genome within 5 years. So far, KOAP has generated 8,004 floxed strains and 9,769 KO (knockout) strains (updated to Oct, 2021). NRCMM also created hundreds of Cre transgenic lines, mutant knock-in models, immuno-deficient models, and humanized mouse models. As a member of the international mouse phenotyping consortium (IMPC), NRCMM provides comprehensive phenotyping services for mouse models. In summary, NRCMM will continue to support biomedical community with new mouse models as well as related services.

Mechanisms of Bone Marrow Failure and Leukemia Progression in Primary Human Fanconi Anemia Stem Cells in Novel FA PDX Model

The goal of this research proposal is to provide better treatments for Fanconi Anemia (FA), an inherited bone marrow failure disorder that affects approximately 1 in 100,000 children. The combination of hematopoietic stress and inherent genomic instability leads to cancer and accumulation of genetic defects is likely the cause of AML progression. We proposed to study primary human cell defective in the FA pathway to delineate pathways of leukemia progression and eventually prevent progression to bone marrow failure or progression to leukemia. Our two aims are to 1)identify molecular vulnerabilities and genetic changes promoting oncogenesis in FA deficient CD34+ cells in vitro and to 2) determine molecular changes at the root of disease progression in primary human FA bone marrow and test potential therapeutic approaches in vivo in MISTRG-kitMUT mice. To achieve this goal we have to i) obtain primary FA patient cells and ii) generate human FANC gene KO CD34+ cells. Note that the COVID pandemic has significantly impaired our progress since 3/15/2020. We have focused our efforts on generating FA defective cells via two mechanisms: a) shRNA mediated knockdown and b) via CRISPR/Cas9 mediated deletion. We have encountered 2 difficulties which we are still addressing: inefficiency of deleting FA genes and selection against deleted cells;silencing of rescue lentiviral vectors in primary hematopoietic cells. With COVID all work had to halt and mouse work was minimal we are expanding colonies and actively transplanting primary FA samples with goal to further optimize engineering of FA samples and transplantation in MISTRG mice.

Animal Models in Human Adenovirus Research.

Human adenovirus (HAdV) infections cause a wide variety of clinical symptoms, ranging from mild upper respiratory tract disease to lethal outcomes, particularly in immunocompromised individuals. To date, neither widely available vaccines nor approved antiadenoviral compounds are available to efficiently deal with HAdV infections. Thus, there is a need to thoroughly understand HAdV-induced disease, and for the development and preclinical evaluation of HAdV therapeutics and/or vaccines, and consequently for suitable standardizable in vitro systems and animal models. Current animal models to study HAdV pathogenesis, persistence, and tumorigenesis include rodents such as Syrian hamsters, mice, and cotton rats, as well as rabbits. In addition, a few recent studies on other species, such as pigs and tree shrews, reported promising data. These models mimic (aspects of) HAdV-induced pathological changes in humans and, although they are relevant, an ideal HAdV animal model has yet to be developed. This review summarizes the available animal models of HAdV infection with comprehensive descriptions of virus-induced pathogenesis in different animal species. We also elaborate on rodent HAdV animal models and how they contributed to insights into adenovirus-induced cell transformation and cancer.

Bridging the B Cell Gap: Novel Technologies to Study Antigen-Specific Human B Cell Responses.

The generation of high affinity antibodies is a crucial aspect of immunity induced by vaccination or infection. Investigation into the B cells that produce these antibodies grants key insights into the effectiveness of novel immunogens to induce a lasting protective response against endemic or pandemic pathogens, such as influenza viruses, human immunodeficiency virus, or severe acute respiratory syndrome coronavirus-2. However, humoral immunity has largely been studied at the serological level, limiting our knowledge on the specificity and function of B cells recruited to respond to pathogens. In this review, we cover a number of recent innovations in the field that have increased our ability to connect B cell function to the B cell repertoire and antigen specificity. Moreover, we will highlight recent advances in the development of both ex vivo and in vivo models to study human B cell responses. Together, the technologies highlighted in this review can be used to help design and validate new vaccine designs and platforms.