Humanized Mice in Infectious Disease Research


Vaccines, HIV, and diseases affecting the human liver are challenging to model without humanization of immune response and creative tissue engineering. Learn more in our guide to using humanized mice in infectious disease research.

Evaluating Vaccines in Humanized Mice

The evaluation of specific antibody production requires an in vivo model; however, species differences limit the utility of experimental animals for this purpose. For example, the lack of interspecies homology between the T cell receptor (TCR) repertoire and major histocompatibility complex (MHC) proteins precludes the evaluation of human vaccine responses in rodents. Humanized mice could be the tool researchers need to bridge that gap. Dr. Yoshie Kametani and a team of pioneering humanized immune system (HIS) model researchers from the Central Institute of Experimental Animals (CIEA) describe a novel model that enables the evaluation of human vaccine responses in mice.

Two common ways to generate immune-system humanized mice involve engrafting either human hematopoietic stem cells (HSCs) or human peripheral blood mononuclear cells (PBMCs) into super-immunodeficient models, like the CIEA NOG mouse®. Super-immunodeficient models encompass a subset of engineered immunodeficient mice harboring unique genetic properties that enhance the engraftment of human cells and tissues.

Limitations of Engrafted Models

HSC-engrafted NOG (huNOG) mice develop a functional human immune system that persists throughout the lifetime of the mouse. However, interspecies differences during immune-system development contribute to suboptimal T cell responses and antibody production in HSC-engrafted models. Therefore, models like huNOG have limited utility for human vaccine evaluation.

By engrafting mature human PBMCs into NOG mice, many of the cross-species impacts on immune system development that affect HSC-engrafted models can be circumvented. Despite this benefit, PBMC-engrafted NOG mice develop xenogeneic graft-versus-host disease (GvHD), which severely limits their lifespan and overall utility for vaccine research.

NOG Variants

To enhance and expand the utility of HIS models, investigators are increasingly turning to NOG variants engineered to express specific human cytokines. For example, huNOG-EXL, an HSC-engrafted NOG model expressing human GM-CSF and IL3, dramatically improves the development of human myeloid lineage cells compared to the non-transgenic huNOG model. Similarly, hIL-2 NOG and hIL-15 NOG express cytokines that specifically enhance human NK cell development from HSCs, making them useful tools for studying human NK cell-mediated antibody dependent cellular cytotoxicity (ADCC).

This second generation of HIS models are beginning to overcome interspecies limitations that impede optimal human immune system engraftment, but novel cytokine transgenic models are still needed. CIEA scientists and pioneers in the HIS-model field have demonstrated that the cytokine-transgenic NOG model expressing human IL-4 (hIL4-NOG) can enable the evaluation of antibody-specific antigen production, illustrating how novel cytokine-transgenic NOG variants can be uniquely important research tools.

A Novel PBMC-Engrafted Model

Despite their potential utility for vaccine evaluation, PBMC-transplanted NOG mice develop GvHD, with onset that depends on donor-specific properties and the initial number of PBMCs engrafted. GvHD onset is also semi-tractable by treatment with specific immune-modulating therapeutics (Figure 1).

Survival curves for PBMC engrafted NOG mice at increasing doses of radiation with, or without pre-treatment with anti-TNFa blocking antibody.
Figure 1: Survival curves for PBMC engrafted NOG mice at increasing doses of radiation
with, or without pre-treatment with anti-TNFa blocking antibody.
Similar to clinical observations of GvHD patients, GvHD in PBMC-engrafted mice has been associated with increased quantities of Th1 cytokines.

Hypothesizing that shifting from Type 1 helper (Th1) to a Th2 phenotype could prevent GvHD while maintaining humoral immunity, CIEA scientists generated transgenic NOG mice expressing hIL4, a representative Th2 cytokine.

Compared to conventional NOG mice, which showed significant weight loss two weeks after PBMC-engraftment, similarly-engrafted hIL4-NOG mice maintained their weight over a twenty-week period of analysis. Thus, GvHD was effectively suppressed in PBMC-engrafted hIL4-NOG mice compared non-transgenic NOG.

When the team of CIEA scientists analyzed the engrafted human T and B cells in hIL4-NOG, they observed dominant CD4+ vs. CD8+ T cell proliferation. Supporting their initial hypothesis, long-term suppression of GvHD was associated with Th2-shifted CD4+ T cells in hIL4-NOG.

Furthermore, conventional memory B cells increased in the PBMC-engrafted hIL4-NOG mice, whereas most of the remaining B cells in the conventional NOG counterparts displayed surface markers indicating B cell exhaustion.

Vaccine Evaluation Performance

In further experimentation, the CIEA team investigated the utility of PBMC-engrafted hIL4-NOG mice for evaluating vaccine effects. Vaccination with a HER2 peptide successfully induced antigen-specific IgG production in PBMC-transplanted hIL4-NOG. Notably, IgG production was independent of the donor's PBMC haplotype, suggesting the HLA haplotype of donor PBMCs may not be relevant to antibody production following immunization.

Collectively, the results suggest that PBMC-engrafted hIL4-NOG mice do not manifest GvHD and can be vaccinated to evaluate peptide-specific IgG antibody production. Moreover, donor HLA status (class II restriction) does not appear to require accommodation in this system.

On a broader scale, these experiments support an important role for novel cytokine transgenic NOG models like hIL4-NOG in advancing important in vivo research capabilities.

Humanized Mice in HIV Research

Thanks to improvements in combined antiretroviral therapy (cART), HIV is no longer a death sentence. But despite decades of investigation, there is no cure for HIV or AIDS.

HIV researchers have been challenged in finding a suitable small animal model, as rodents such as mice and rats cannot be infected by HIV. Existing animal models such as chimpanzees are not ideal as they are expensive, more challenging to work with and have a higher ethical burden for use. Furthermore, while some non-human primates (NHPs) can be infected with HIV, they do not develop similar disease as in humans.

Can humanized mice speed up HIV drug discovery, further improving the quality of life for those living with HIV?

Mice with Human Immune Systems Fill a Critical Gap

A related virus, SIV, is thus typically used to model HIV in NHPs. In this challenging environment, mice with human immune systems fill a critical gap, serving as a small animal model which can be infected with HIV and model the disease. In the Foreword to the textbook Humanized Mice for HIV Research, Susan Swindells says, "Humanized mice have evolved into an invaluable alternative to SIV-based nonhuman primate models, as they are simpler, less costly, and also highly susceptible to HIV infection."

Modeling of HIV in Mice

Modeling of HIV in mice engrafted with human immune cells got its start in the late 1980's with the development of the scid-hu thy/liv model1 by Mike McCune. The field has made huge advances since then, with the development of super immunodeficient mice such as the CIEA NOG mouse® contributing significantly to the development of better models of the human immune system.

Humanized Mice for HIV Research

Humanized Mice for HIV Research is a comprehensive overview of human immune engrafted mouse models as they apply to HIV, but also with relevance to other fields of study. Chapters contributed by Mamoru Ito, Mike McCune and Lenny Shultz introduce the history of immune engrafted mice. A chapter by Paul Denton, Tomonori Nochi and J. Victor Garcia has an interesting discussion of the NOD scid relative to NOG/NSG models for immune engraftment. In terms of reconstitution of intestinal lymphoid tissues, the NOD scid offers some key advantages. The function of human cells reconstituted in immune deficient hosts is explored by several contributors, with chapters covering T, B and NK cells as well as antigen presenting cells such as macrophages and dendritic cells.

Modelling HIV Transmission

Some of the most interesting work using immune engrafted mice to model HIV has been transmission studies. These models can be used to study oral, vaginal and rectal transmission of HIV, and they have been used to study many different types of prophylactic approaches. Immune engrafted mice have also been used to model HIV latency and test methods of overcoming such latency. A section of the book is devoted specifically to HIV therapeutic development; this includes practical discussions of species differences in pharmacokinetics, pharmacodynamics and drug distribution. Although the work is titled Humanized Mice for HIV Research, this book also includes a section on modeling other human specific/selection pathogens in human immune system mice. Pathogens covered include dengue, Epstein-Barr virus, HTLV-1, malaria and others.

Humanized Mice for HIV Research is an excellent reference, not just for HIV researchers, but also for anyone interested in working with immune engrafted models. The sections on characterization of the models provide valuable insights applicable to studies in other areas such as tumor immunology. As Victor Garcia says in the concluding chapter, "The availability of these models has completely transformed the landscape in the fields of human immunology and infectious diseases...More recent uses in other areas are beginning to demonstrate the utility and the promise of great future potential applications."

Preclinical HIV Research Webinar

To support HIV research, Taconic Biosciences hosted an HIV Drug Discovery webinar with Dr. Patrick Nef (CEO) and Dr. Sebastien Tabruyn (CSO) of TransCure BioServices.

TransCure is a contract research organization (CRO) providing support to the pharmaceutical and biotechnology companies through preclinical candidate selection, immunosafety, and efficacy in humanized mouse models related to inflammation, oncology, and infectious diseases.

In this exclusive Taconic webinar, Drs. Nef and Tabruyn discuss in detail how a humanized NOG mouse could be an in vivo model for HIV drug discovery, characterize anti-HIV prevention strategies, and investigate novel HIV eradication strategies.

Unlike other models, human immune cells including T cells are present in humanized NOG mice. As reported by TransCure, stable and elevated HIV viral load in the blood can be observed without virus modification in human immune system engrafted NOG mice such as the huNOG. As no GvHD is observed and hCD45 chimerism is stable in humanized NOG, huNOG can be used for HIV latency and resistance study beyond 120 days.

TransCure's Guideline for Use of huNOG in HIV Research

Study Design:
  • Protective/vaccine prime, anti-HIV efficacy/combotherapy, rebound/latency, and resistance can be studies in humanized NOG.
HIV Strain:
  • Both cytopathic and cytolytic strain can be studied
    • TransCure discussed two HIV-1 strains in the presentation: R5/yu2 and X4/NL4-3 strains.
  • Tropism: CCR5, CSCR4, or combined
Viral Load:
  • ~106 copies/ml has been observed
Route of Infection:
  • IV, IP, and vaginal infections are possible
Route of Treatment:
  • IV, IP, gavage, food pellet/drinking water has been tested
Endpoints/disease Progression:
  • HIV viral load in blood by qPCR
  • Leukocyte population by flow cyometery
  • Survival rate and weight loss
Study Length:
  • Long term resistance study is possible: ~120 days data were shown in the presentation.
HARRT (Highly Active Antiretroviral Therapy):
  • Combination of raltegravir, lamivudine, tenofovir (food pellet at libidum) were used in the data shown.

HIV-Associated Liver Disease in Dual-Humanized TK-NOG Mice

A recent study by Dagur et al. describes a novel method for studying the human liver in the context HIV-1 infection, using a dual reconstituted humanized mouse model based on the TK-NOG1.

Humanized mice are now considered a viable alterative to primate models in studying HIV infection2,3. Amongst these are CIEA NOG mice and NSG mice engrafted with human immune cells, including CD34+ hematopoietic stem cells (HSC) and peripheral blood mononuclear cells (PBMC). HSC-engrafted models have the advantage of stable, long-term engraftment, but, unlike PBMC-engrafted models, HSC-engrafted models are generally limited to T cell lineages. Each of these models can be productively infected with HIV-1 and have been widely used to study HIV viral dynamics and new ARV therapies3.

Modelling the Human Liver

An obvious limitation to these models is the absence of a human liver. Bone marrow/liver/thymus (BLT)-engrafted mice help to overcome this limitation; however, these animals are technically challenging to produce and interference with the native mouse liver can obscure the effects of engrafted human hepatocytes. The TK-NOG model allows for orthotopic transplantation of human hepatocytes into the mouse liver, while simultaneously ablating mouse hepatocytes due to a thymidine kinase transgene expressed under the mouse albumin promoter4.

The TK-NOG has been used extensively to study human liver drug metabolism and toxicity. Additionally, TK-NOG mice can be engrafted with human HSCs to produce a dual reconstituted humanized liver/immune system mouse model.

Studying HIV- and Drug-Induced Liver Pathology

Dagur et al. used this dual reconstituted TK-NOG model to study how HIV-1 infection affected the human liver. They showed that chronic HIV-1 infection (16 weeks) induced hepatocyte damage in these dual reconstituted mice, but not in mono reconstituted mice, or in HIV-uninfected dual reconstituted mice. This effect was associated with a decrease in human albumin production, a proinflammatory milieu in the liver, and a decrease in liver CD4+ T cells. Thus, the TK-NOG offers a novel model to study HIV-induced human liver pathogenesis.

Drug-Induced Hepatotoxicity

While liver pathology has been described in patients living with unsuppressed viral loads, the advent of highly-active ARV and long-term viral suppression has largely replaced HIV-induced with drug-induced hepatotoxicity. This dual reconstituted TK-NOG model offers a model to simultaneously monitor ARV efficacy, along with human liver metabolism and toxicity, all within the context of HIV-1 infection and subsequent viral suppression.

Metabolism of HIV Therapies

A related study recently reported the development of a novel NOG strain that also allows for human metabolism of ARV drugs. This model expresses the human drug metabolizing enzymes CYP3A4/7 and the nuclear receptors PXR and CAR in a NOG background5. The authors showed that this model recapitulated human specific ARV metabolism in the mouse liver, suggesting that this model might also help predict ARV metabolism and efficacy following HSC engraftment and HIV-1 infection.

Modelling Malaria with Humanization and Tissue Engineering

The fight against malaria is hampered by the lack of good animal models. The most dangerous malaria parasite, Plasmodium falciparum, has a complex life cycle involving two hosts: mosquitoes and humans. In humans, the parasite has both a liver stage and a red blood cell stage.

Because of its host species specificity, P. falciparum has been challenging to model in vivo, particularly during the liver stage.

Humanized Mouse Models of Malaria

One promising approach to modeling the liver stage of malaria parasites is to use mice with humanized livers. An immunodeficient mouse, engineered with susceptibility to liver injury, can be depopulated of mouse hepatocytes and engrafted with human hepatocytes which repopulate the mouse liver.

The TK-NOG mouse is one such model. As with HIV-related liver disease, these mice, engrafted with human hepatocytes, can be used to model the malaria liver stage. But generation of chimeric liver mice is expensive, difficult, and requires access to specialized mouse strains.

Modeling Malaria with Artificial Livers

Ng et al. report on a new approach to this problem via the use of engineered artificial livers implanted into readily available standard immunodeficient strains, such as nude mice.

The researchers developed porous human ectopic artificial livers (p-HEALs) via tissue engineering and implanted them successfully into the intraperitoneal space of NCr nude mice. In a proof of concept experiment, the team demonstrated that p-HEALs implanted into nude mice could be infected with P. falciparum in vivo.

The authors argue the tissue engineering approach may be more efficient and scalable compared to established chimeric liver models. As a next step, the researchers contemplate a dual humanized mouse with p-HEALs and a human immune system to study host-parasite interactions.

References:
1. McCune JM, Namikawa R, Kaneshima H, Shultz LD, Lieberman M, Weissman IL.(1988) The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science. 241(4873):1632-9.
2. Watanabe S, Terashima K, Ohta S, Horibata S, Yajima M, Shiozawa Y, Dewan MZ, Yu Z, Ito M, Morio T, Shimizu N, Honda M, Yamamoto N. Hematopoietic stem cell-engrafted NOD/SCID/IL2Rgamma null mice develop human lymphoid systems and induce long-lasting HIV-1 infection with specific humoral immune responses. Blood. 2007 Jan 1;109(1):212-8.
3. Satou Y, Katsuya H, Fukuda A, Misawa N, Ito J, Uchiyama Y, Miyazato P, Islam S, Fassati A, Melamed A, Bangham CRM, Koyanagi Y, Sato K. Dynamics and mechanisms of clonal expansion of HIV-1-infected cells in a humanized mouse model. Sci Rep. 2017 Jul 31;7(1):6913.
4. Hasegawa M, Kawai K, Mitsui T, Taniguchi K, Monnai M, Wakui M, Ito M, Suematsu M, Peltz G, Nakamura M, Suemizu H. The reconstituted 'humanized liver' in TK-NOG mice is mature and functional. Biochem Biophys Res Commun. 2011 Feb 18;405(3):405-10.
5. McMillan JM, Cobb DA, Lin Z, Banoub MG, Dagur RS, Branch Woods AA, Wang W, Makarov E, Kocher T, Joshi PS, Quadros RM, Harms DW, Cohen SM, Gendelman HE, Gurumurthy CB, Gorantla S, Poluektova LY. Antiretroviral Drug Metabolism in Humanized PXR-CAR-CYP3A-NOG Mice. J Pharmacol Exp Ther. 2018 May;365(2):272-280.