Humanized mice in infectious diseases

Accepted Manuscript Title: Humanized mice in infectious diseases Author: W. Ernst PII: DOI: Reference:

S0147-9571(16)30082-0 http://dx.doi.org/doi:10.1016/j.cimid.2016.08.006 CIMID 1090

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Title Page:

Type of Article: Review Article

Title: Humanized mice in infectious diseases Authors: Ernst W1* 1

Clinic of Gynecology and Obstetrics St. Hedwig, University of Regensburg,

Regensburg, Germany

*

Corresponding author

Dr. Wolfgang Ernst Clinic of Gynecology and Obstetrics St. Hedwig Regensburg, Bavaria; Germany Phone: +49(0)941 944 8912 Email: [email protected]

Title: Humanized mice in infectious diseases

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Highlights

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Advantages of the humanized mouse as an animal model The humanized mouse model in viral infections The humanized mouse model in bacterial and parasitic infections Methods to improve the animal model

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Manuscript Organization:

Abstract: The pathogenesis of infectious agents with human tropism can only be properly studied in an in vivo model featuring human cells or tissue. Humanized mice represent a small animal model featuring human cells or tissue that can be infected by human-specific viruses, bacteria, and parasites and also providing a functional human immune system. This makes the analysis of a human immune response to infection possible and allows for preclinical testing of new vaccines and therapeutic agents. Results of various studies using humanized mice to investigate pathogens with human tropism are presented in this review. In addition, the limitations of humanized mice and methods to improve this valuable animal model are discussed.

Keywords: Review; humanized mice; infectious diseases; human specific pathogens

Abbreviations: WHO: World Health Organization; TNF: tumor necrosis factor; IL: interleukin; HSC: hematopoietic stem cell; NSG: NOD/SCID/γc−/−; NK: natural killer; DC: dendritic cell; TCR: T cell receptor; DTH: delayed type hypersensitivity; HLA: human leukocyte antigen; Ig: immunoglobulin; HIV: human immunodeficiency virus; APOBEC3: apolipoprotein B mRNA editing enzyme catalytic polypeptide 3; siRNA: small interfering RNA; shRNA: small hairpin RNA; CCR5: C-C chemokine receptor type 5; EBV: Epstein-Barr virus; IFN: interferon; GM-CSF: granulocyte-macrophage colonystimulating factor; HBV: hepatitis B virus; HCV: hepatitis C virus; HCMV: human cytomegalovirus; G-CSF: granulocyte-colony stimulating factor; DENV: Dengue virus; HTLV-1: human T cell leukemia virus type 1; HSV-2: herpes simplex virus type 2; HuNoV: human norovirus; JCV: John Cunningham virus; P.: Plasmodium; SCID: severe combined immunodeficiency; L.: Leishmania; N.: Neisseria; S.: Salmonella; MIP: macrophage inflammatory protein; B.: Borrelia; S.: Streptococcus; GBS: group B Streptococcus; CLP: cecal ligation and puncture; HMGB1: high-mobility group protein 3

B1; M-CSF: macrophage colony-stimulating factor; ICAM-1: intercellular adhesion molecule 1; SIRP: signal-regulatory protein; SCF: stem cell factor; NOD: non-obese diabetic; MHC: major histocompatibility complex; Rag: Recombination activating gene; Tat: trans-activator of transcription, Rev: regulator of expression of virion proteins; FAH: Fumarylacetoacetase; uPA: urinary plasminogen activator; tg: transgenic; Alb: Albumin; qPCR: quantitative polymerase chain reaction

Introduction

According to the World Health Organization (WHO), infectious diseases are among the leading causes of death worldwide [1]. They are responsible for the deaths of more than two thirds (68%) of children younger than 5 years, and had a worldwide death toll of 5.97 million individuals in 2008 [2]. These facts demonstrate the importance of research in the field of infectious diseases, in order to gain new insights that lead to a better understanding of infectious agents and pathogenesis of the infection as well as to develop and test new therapeutic agents and vaccines aiming to reduce the death toll conferred by infectious diseases. Due to the fact that infection and the resulting immune response are complex processes, in vitro models are only suitable to a limited extent. Parameters like pattern and kinetics of pathogen dissemination, migration of leukocytes and disease progression among others cannot be studied in vitro. Also, efficacy of vaccines and therapeutic agents can only be tested in vivo. Furthermore, only in vivo models are able to predict adverse reactions like toxicity of drugs and their metabolic products.

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Small animal models such as mice and rats are frequently used for biomedical research for several reasons. They are inexpensive, easy to breed and maintain, have a short generation time, and can easily be handled and restrained due to their docile nature. Even though the mouse genome is smaller than the human genome due to less repetitive DNA sequences, we share approximately 97.5% of our working DNA with these animals [3]. In addition, many studies documented that mice are suitable models for infections, and particularly sepsis, since they show a variety of symptoms, display similar responses, and adequately emulate the human disease. In both species IL-6 is a biomarker for sepsis mortality, immune and gastrointestinal cells become apoptotic, and autophagy in tissues can be observed [4]. Therefore, mice are commonly used as animal models to study infectious diseases and test therapeutic agents. However, the usefulness of mice as a suitable model for human physiology is still under debate. Arguments against the use of mice are for example the fact that certain drugs and therapies failed in human trials even though they worked in murine models. One example is Fialuridine, an experimental hepatitis B drug, which led to the death of five patients in a clinical trial due to liver toxicity. This severe side effect has not been seen in the previous animal studies [5]. Other examples are various therapies (e.g. anti-tumor necrosis factor (TNF) monoclonal antibodies, soluble TNF receptors, interleukin-(IL) 1ra) which worked in murine sepsis models but failed in subsequent clinical trials [6,7]. The immunomodulating anti-CD28 monoclonal antibody TGN1412 induced life threatening allergic reactions in all participating test subjects in a clinical trial but caused no serious side effects in previous animal tests [8]. The reasons for the diverging effects in mice and humans seem to be based on a variety of differences in the immune system 5

between the two species with at least 67 known discrepancies [9] as well as a generally different genomic response to inflammatory disease [10]. One could argue that animal models failed to predict the outcome in humans in certain cases and conclude that animal models have no predictive value for human diseases [11]. This, however, would ignore the fact that animal research played - and still plays - a significant part in basic biomedical research. Animal models were also vital for medical breakthroughs in the last decades and most Nobel Prizes in Physiology or Medicine were awarded to findings based on animal research. Currently there is basically no viable alternative to animal models in biomedical research in general and research on infectious diseases in particular. Instead of criticizing current animal models, we should rather focus on improving and refining them. A good example for such an improved animal model is the so called ‘humanized’ mouse. Humanized mice are defined in this review as immunodeficient mice which have been engrafted with human cells and/or tissue (usually hematopoietic stem cells (HSC) and tissue like fetal liver and thymus). Engrafted human HSC give rise to a complete and functional human immune system. There are high variances in the level of engraftment concerning composition as well as functionality of the human immune system, depending on the immunodeficient mouse strain and the technique used for humanization [12,13]. The generation of humanized mice and composition and function of the resulting human immune system in these animals will be described using the example of the NOD/SCID/γc−/− (NSG) strain engrafted with human HSC by neonatal injection. NSG mice are irradiated and subsequently transplanted (via intrahepatical, intracardiac or facial vein injection) with human HSC. The HSC can be obtained from different sources 6

(umbilical cord blood, mobilized HSC from adult donors, or aborted fetuses). Within 8 to 12 weeks, a functional human immune system develops since human HSC give rise to granulocytes, monocytes/macrophages, dendritic cells (DC), natural killer (NK), T and B cells and even erythrocytes and platelets. In addition, lymphocyte subsets are generated. These include myeloid and plasmacytoid DC as well as CD4+ CD8- T helper cells, CD4CD8+ cytotoxic T cells and even regulatory T cells. T cells develop in the murine thymus through the expected stages (CD4- CD8- to CD4+ CD8+ to either CD4+ CD8- or CD4CD8+). T cells in humanized mice display a complex T cell receptor (TCR) repertoire, human leukocyte antigen (HLA)-dependent cytotoxicity, mount a delayed type hypersensitivity (DTH) response and proliferate after stimulation. The immune system of humanized NSG mice also features subpopulations of NK cells (NKp46+ CD56−, CD56bright CD16− KIR− and CD56dim CD16+ KIR+ cells) which possess cytotoxic capabilities, degranulate and produce interferon γ upon stimulation. B cells produce antigen specific IgM and are also able to undergo class switching to IgG [12, 14-17]. Since the human immune system in humanized mice features all leukocyte subsets and possesses functional capabilities, a variety of studies on human pathogens and infectious diseases have been performed using this animal model. This review will give an overview over these studies.

Viral infections

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Certain viruses are specific to humans as they require human cells for infection (e.g. leukocytes), replication and pathogenesis which are absent in regular animal models. The humanized mouse is a small animal model which can be successfully engrafted with a variety of human cell types and/or tissue and is therefore a suitable and very valuable tool to investigate the diseases caused by human-specific viruses and also to test new therapies and vaccines. This is the reason why several studies have been performed using this animal model (see table 1). However, this review will not extensively discuss viral infections in humanized mice, since reviews on this topic already exist, especially for the human immunodeficiency virus (HIV) [18-22].

The most intensively studied virus in humanized mice is HIV. In the majority of studies, especially the early ones, the animals were infected by intravenous or intraperitoneal injection. However, since humanized mice boast a human mucosal immune system, they were also successfully infected through vaginal, rectal, and oral transmission, which are the common routes for human infection [23-26]. Not only can humanized mice be effectively infected with HIV, they additionally feature major hallmarks of the HIV infection and pathogenesis in humans. After entry through the mucosa, target cells infected with HIV serve as a vehicle for dissemination to lymphoid tissue and subsequent systemic infection. Via infected macrophages, the virus is able to cross the blood brain barrier leading to viral neuropathogenesis which can also be seen in humans [27,28]. Similar to humans, major sites of virus replication are thymus, spleen and lymph nodes. Infected animals developed high levels of viremia, marked CD4+ T cell loss in blood and lymphoid organs, and also sustained long-term HIV infection [20]. HIV latency via 8

infection of resting CD4+ cells which serve as a latent reservoir protecting the virus from antiretroviral therapy can be observed in humanized mice as well [29,30]. Infection of the animals led to the production of HIV-specific antibodies and to a cytotoxic T cell response [31]. When humans are infected with HIV, an evolution of the viral genome occurs during the course of the infection. Especially the envelope gene env is affected by this mechanism. Ince et al. could show that this evolution of the env gene, which is driven by selective pressure of the immune system, also occurred in humanized mice [32]. Humanized mice not only displayed an adaptive immune response, but also mounted a functional innate immune response against HIV via apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3) which effectively restricts the virus [33]. Since the humanized mouse has been shown to be a suitable model for HIV infection, it has been used to test the efficacy of anti-HIV drugs. Studies have tested the efficacy of already approved and applied drugs like emtricitabine and tenofovir disoproxil fumarate in humanized mice. Prophylactic treatment with these antiretroviral drugs prevented vaginal, rectal and intravenous HIV transmission in the animals [24-26, 34]. These results correspond well with findings in human subjects [35] and demonstrate the usefulness of the humanized mouse in testing novel therapeutic agents due to the good transferability of the results from this animal model to human patients. Hence, experimental drugs like the strand transfer inhibitor L-870812 or a tat peptide inhibitor have been tested using this animal model [36,37]. Established anti-HIV therapies and drugs are effective in humanized mice. They also represent a suitable model to test experimental drugs. In addition, this animal model can be used to find new therapeutic approaches. This has been shown by successfully testing 9

a novel form of therapy relying on genetic modification in humanized mice. This new therapeutic approach used small interfering RNA (siRNA) or small hairpin RNA (shRNA), leading to the downregulation of the expression of viral genes (e.g. tat) [38], or essential proteins for viral entry (e.g. CCR5) [39]. Another novel therapy was tested in a study by O’Connell et al. showing that expression of human IL-7 increased the number of T lymphocytes but not the viral load in humanized mice [40]. The administration of IL-7 could have the same effect in HIV patients and should therefore be investigated further.

Besides HIV, Epstein-Barr virus (EBV) has also been studied extensively using the humanized mouse model [41]. EBV is a herpesvirus and has a high infestation rate. The life-long infection that humans develop is usually asymptomatic. However, the virus is associated with Hodgkin's lymphoma, Burkitt's lymphoma as well as other forms of cancer such as nasopharyngeal carcinoma. It also increases the risk of developing autoimmune diseases such as rheumatoid arthritis. After inoculation with EBV, a persistent infection and B cell lymphoproliferative disease develops in humanized mice. The immune system of the animals mounts a T cell response and produces EBV-specific IgM [42]. The T cell response is HLA restricted and protective since T cell depletion led to an increase in the viral titer and virus-associated lymphoproliferative disease [43]. Humanized mice can also serve as a model for virusassociated hemophagocytic lymphohistiocytosis since infiltration of activated cytotoxic T cells

into

organs,

interferon-

(IFN)

γ

cytokinenemia,

normocytic

anemia,

thrombocytopenia as well as high plasma levels of EBV-encoded small RNA1 can be observed upon infection with EBV corresponding well with the findings in patients [44]. 10

Kuwana et al. generated a model using humanized mice featuring a similar clinical picture as rheumatoid arthritis upon EBV infection [45]. The animals developed erosive arthritis, synovial membrane proliferation, infiltration of lymphocytes (B cells, CD4+ and CD8+ T cells) as well as macrophages, pannus formation and bone marrow edema. The humanized mouse model has also been used to test the efficacy of novel experimental treatments for EBV-associated lymphoproliferative disease. Low doses of IL-2 prevented EBV-associated lymphoproliferative disease in humanized mice which can occur in patients with AIDS [46, 47]. Protection against this disorder was also achieved using granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-2 [48]. A combination of Rituximab and IL-2 was successfully tested in a human B-cell nonHodgkin's lymphoma model. This humanized mouse model also enabled Eisenbeis et al. to find the optimal treatment regimen [49]. Adoptive cellular immunotherapy using plasmacytoid dendritic cells loaded with EBV-derived peptides induced a robust EBVspecific cellular immune response in humanized mice with EBV-associated lymphoproliferative disease [50].

The hepatitis B virus (HBV) and hepatitis C virus (HCV) establish persistent infections causing liver disease such as cirrhosis, steatosis, and hepatocellular carcinoma in humanized mice. Since HBV and HCV infect liver cells, the animals need to be engrafted not only with human HSC but also with human hepatocytes to study the viral infection and pathogenesis [51]. After co-transplantation of human HSC and human liver progenitor cells into immunodeficient mice infected with HCV, the human immune system generated a virus-specific, cell-mediated immune response and the animals 11

developed liver inflammation, hepatitis, and fibrosis [52,53]. Not only can the animal model be used to study the pathogenesis of the HCV infection and the response of the human immune system, but also to test drugs and therapies as recent studies have shown. Vercauteren et al. used the humanized mouse model to show that using an entry inhibitor in combination with anti-HCV direct-acting antiviral therapy prevents viral breakthrough by drug-resistant HCV mutants. These HCV mutants developed in animals receiving standard antiviral therapy and also pose a problem in human patients [54]. Humanized mice were also used to show that injection of monoclonal antibodies against the HCV coreceptor - scavenger receptor class B type I - not only has a protective effect in case of subsequent HCV challenge but is also able to clear HCV from the circulation of infected animals [55]. Immunoprophylaxis using viral vectors to deliver neutralizing antibodies also protected humanized mice from HCV infection [56]. The same animal model used for HCV infection was utilized to analyze the pathogenesis of HBV infection. Not only did humanized mice exhibit liver inflammation and fibrosis after HBV infection, the animals also showed infiltration of high levels of M2-like macrophages in the liver, which is consistent with findings in human patients [57].

Human cytomegalovirus (HCMV) is a herpesvirus and poses a serious problem for recipients of organ and stem cell transplants from seropositive donors. Humanized mice have successfully been infected with HCMV, and the animal model displayed latent viral infection of human cells of the hematopoietic linage as well as viral dissemination into liver, spleen and bone marrow [58-60]. Using this animal model, Smith et al. could observe reactivation of latent HCMV in monocytes/macrophages after G-CSF treatment. 12

These results indicate that the widely used method of mobilization of HSC and granulocytes via G-CSF is likely to increase the risk of HCMV transmission if the donor is seropositive [58]. Kawahara et al. generated a model to study HCMV infection of hepatocytes in context with human liver NK cells by cotransplanting both cell types into immunodeficient mice. In this model, the human NK cells were able to control the HCMV infection, which indicates, that this model could be used to analyze HCMV infection in hepatocytes [61].

Dengue virus (DENV) is usually transmitted via bites of infected mosquitos and can lead to dengue fever, dengue hemorrhagic fever and dengue shock syndrome. For more detailed information about the virus, disease and animal models of dengue infection please see Zompi & Harris [62]. Humanized mice can be successfully infected with DENV [63-66]. These animals had a decreased platelet count, fever and erythema which are clinical signs of dengue fever. Humanized mice developed high viremia and dissemination of the virus into bone marrow, spleen, liver and blood. Cos et al. developed a very realistic infection model by allowing infected mosquitos to bite humanized mice, which resulted not only in DENV infection but a more severe form of the disease with higher viremia, erythema, and thrombocytopenia compared to animals that were inoculated via injection of the virus [67]. The immune system of humanized mice mounted a humoral immune response with anti-DENV IgM and IgG. HLA-transgenic animals even responded with a functional DENV-specific human T cell response after infection [68] making the humanized mouse a suitable model to study the pathogenesis of the infection and test vaccines as well as novel therapies. Sridharan et al. used humanized 13

mice transplanted with human HSC from fetal liver to determine the mechanism of virusinduced thrombocytopenia caused by DENV infection. He found that in infected animals the decrease in platelets stemmed from a reduction of human megakaryocytes and megakaryocyte progenitors in the bone marrow [69]. The humanized mouse model has also been successfully used to test the efficacy of experimental antiviral drugs. In a study by Frias-Staheli et al. the adenosine nucleoside inhibitor NITD008 effectively reduced the viral titer in DENV infected animals [70].

The human T cell leukemia virus type 1 (HTLV-1) is associated with adult T cell lymphoma (ATL) and other diseases. Different humanized mouse models have been used to study HTLV-1 pathogenesis [71]. In recent studies Tezuka et al. generated a humanized mouse model that developed adult T-cell leukemia and featured a HTLV-1specific adaptive immune response [72], and Saito et al. developed an animal model where he successfully tested a prophylactic anti-HTLV-1 antibody treatment which protected the mice from infection [73].

There are a number of studies on other viral pathogens using the humanized mouse as a suitable animal model. These pathogens will not be discussed in detail, since this would go beyond the scope of this review. Kwant-Mitchell et al. could show that humanized mice generate both virus-specific innate and adaptive immune responses after infection with herpes simplex virus type 2 (HSV-2) [74]. Taube et al. successfully infected humanized mice with human norovirus (HuNoV) and hence generated a model to study HuNoV infection in context with a human immune system [75]. A humanized mouse 14

model to study John Cunningham virus (JCV) infection was established by Tan et al [76]. In this study, animals inoculated with JCV developed both cellular and humoral immune responses against the virus.

Bacterial and parasitic infections

While parasitic and bacterial pathogens are generally less host specific compared to viruses, certain exceptions do exist. Most importantly, animal models often fail to mimic the human pathogenesis of a disease and the immune system of humans and animal models differ in certain aspects which might considerably impact on the effect of drugs and vaccines. This is the reason why a number of studies have been performed using humanized mice as an animal model (see table 2).

Plasmodium falciparum is the most lethal Plasmodium parasite causing malaria in humans posing a major threat to global health. Research on this pathogen and developing new antimalarial drugs have proven difficult because the parasite has a complex life cycle and a tropism for human cells, especially hepatocytes. Since the parasite has developed resistances against proven drugs, there is definitely a need for new animal models for the development of new drugs against malaria. For more information on P. falciparum and various models used to study the parasitic infection see Vaughan et al 2012 [77]. A model with P. falciparum blood-stage infection using humanized mice was generated by Jimenez-Diaz et al. [78]. Here NSG mice were stably engrafted with P. falciparum 15

infected human erythrocytes via intraperitoneal injection. The infected animals were used to assess the efficacy of the antimalarial drugs artesunate, chloroquine, pyrimethamine, and the novel experimental drug GSK932121 against different strains of malaria. The drugs showed a clear dose response and effective neutralization of non-resistant strains. These results indicate that this model could be useful in preclinical testing of novel drugs. By engrafting NSG mice with human erythrocytes prior to injecting P. falciparuminfected erythrocytes, using the intravenous route for inoculation and treating the animals with immunosuppressive agents to dampen the murine innate immune system, Arnold et al. improved the prior humanized mouse model [79]. This modified model features consistent, long lasting and high parasitemia in all infected mice. Furthermore it also supported the development of sexual stages of P. falciparum. Due to the complex life cycle of P. falciparum, the parasite requires not only human erythrocytes, but also human hepatocytes for its complete development. Sacci et al. generated a humanized mouse model to study the liver stages of the parasite by injecting human hepatocytes into immunodeficient SCID Alb-uPA mice [80]. The P. falciparum sporozoites that were injected into the animals developed to mature liver stage schizonts containing merozoites which possessed the ability to infect human erythrocytes. A similar model has been used to test different forms of infection and to develop a method for quantification of the parasite load in animals using qPCR to be able to test the efficacy of drugs and other compounds against the liver stages of P. falciparum [81].

The Leishmania parasite causes leishmaniasis, a disease which affects 12 million people worldwide. Due to a lack of vaccination and the fact that current therapeutic agents have 16

serious side effects, an animal model which can be used to study experimental drugs and vaccines would be very useful. Humanized mice have been successfully infected with Leishmania major [82]. The animals generated a L. major-specific T cell response and parasites invaded human macrophages at the site of infection and in lymphoid organs. However, the human immune system was unable to control the infection, and the mice died after systemic dissemination of L. major. Treatment of infected animals by oral administration of Miltefosin reduced the parasite load but simultaneously induced liver damage and weight loss. Since these are common side effects in human patients, the humanized mouse could be a useful tool for research and preclinical testing of novel antileishmaniasis drugs.

The bacterial pathogen Neisseria meningitides is responsible not only for meningitis but also for septic shock which has a high mortality rate. Humanized mice represent a suitable animal model to study N. meningitides infection since the pathogen has a human tropism and is dependent on human cells for adhesion and human transferrin to scavenge iron [83]. Melican et al. generated a humanized mouse model to study N. meningitides infection and vascular damage by engrafting human skin onto immunodeficient SCID/Beige mice [84]. The animals received human transferrin and were infected via intravenous injection of N. meningitides. The bacteria adhered to the endothelium of the human skin resulting in the release of pro-inflammatory cytokines by the human cells leading to neutrophil infiltration and inflammation. The skin grafts also showed vascular damage with thrombosis, hemostasis, and vascular leakage. Some animals even

17

developed purpura. This pathology closely resembles that of patients, indicating that this animal model could be a useful tool to study the disease and test new therapeutic agents.

Salmonella enterica serovar Typhi (Salmonella Typhi) is a bacterial pathogen with human tropism and the cause of typhoid fever. Although vaccines exist, their efficacy is not very high, and the rise of drug-resistant strains is cause for concern since there are 21 million cases of typhoid fever each year [85]. Several groups have generated humanized mouse models for S. Typhi infection by engrafting immunodeficient mice with human HSC [86-88]. Humanized mice not only got infected with S. Typhi but showed dissemination of the pathogen into spleen and liver as well as replication in these organs. The animals developed pathological changes in liver (Kupfer cell swelling) and spleen (granulomas) which are also common in human patients [86]. Elevated levels of human TNFα and IL-6 were caused by a systemic immune response to the shed lipopolysaccharide of S. Typhi, and are also present in human typhoid fever [85]. Other pro-inflammatory cytokines (IL-8, IL-12, IFNγ), chemokines (MIP-1α, IP-10) and the anti-inflammatory cytokine IL-10 were also elevated in the infection model of Song et al., indicating an innate immune response against the pathogen [87]. Some of the animals mounted an adaptive immune response by generating S. Typhi-specific antibodies [87]. Firoz Mian et al. detected neurological signs of meningitis, which is a rare complication in S. Typhi infections in humans [85,88], and spreading to, as well as replication of the bacteria in liver, spleen, blood, and bone marrow. These results indicate that the humanized mouse might be a promising tool to study the pathogenesis of S. Typhi infection and test vaccine and drug candidates. 18

Relapsing fever is a disease caused by Borrelia hermsii which are transmitted via bites from infected ticks. Although murine models for relapsing fever exist, the disease progression and the resolution in humans are not fully understood yet [89]. Vuyyuru et al. successfully infected NSG mice engrafted with human HSC via intraperitoneal injection [89]. The animals were able to effectively control bacteremic episodes of different B. hermsii strains by mounting a pathogen-specific IgM response. The disease progression and the antibody response in humanized mice is similar to human relapsing fever making this animal model a valuable tool to study the cellular and molecular mechanisms of the disease.

The humanized mouse model has also been used as a neonatal Streptococcus agalactiae(group B Streptococcus, GBS) sepsis model. Despite intensive care and prophylactic antibiotic treatment, S. agalactiae remains a significant threat to neonates in the western world [90]. In this study, neonatal NSG mice engrafted with human HSC were infected with GBS via intraperitoneal injection [90]. After infection a systemic dissemination of the pathogen was observed and animals inoculated with moderate to lower doses of GBS were able to clear the infection over time in essentially all organs investigated (spleen, kidney, liver, lung, brain, bone marrow and peritoneum). Upon infection, the human immune system reacted with production of pro- and anti-inflammatory cytokines and chemokines (TNFα, IFNγ, IL-1β, IL-6, IL-8, IL-10) and leukocyte trafficking. The effect of the drugs Betamethasone and Indomethacin which are given in case of sepsis-induced premature birth to promote lung maturation and delay birth were also assessed. The drugs 19

altered human B, T, and myeloid cell populations in blood, bone marrow and spleen but only Betamethasone treatment resulted in a higher systemic bacterial burden. In summary, the humanized mouse model gave new insights on the impact of Betamethasone and Indomethacin on the human immune system and its functions during GBS infection/sepsis.

Sepsis not only poses a prominent threat to neonates, but is the leading cause of death in intensive care units. An important aspect of sepsis is that the mortality rate is still high and remains unchanged in the last 25 years [16]. A major problem in sepsis research is the fact that a number of treatments which were effective in animal models of sepsis failed in clinical trials [6,7,16]. Unsinger et al. generated a sepsis model using humanized mice [16] because of the differences between the immune system of animals - especially mice - and humans [9]. NSG mice were engrafted with human HSC and an intraabdominal peritonitis (sepsis model) was induced by perforation of the cecum (cecal ligation and puncture, CLP). The peritonitis induced apoptosis in B and T cells and the animals showed markedly increased pro- (TNF-α, IFN-γ IL-6) and anti-inflammatory (IL-10) cytokine levels. Both findings are typical for septic human patients. Ye at al. used a similar humanized mouse model to analyze the pathogenesis of sepsis and to test a novel siRNA treatment [91]. In this animal model, high levels of human cytokines (TNFα, IL-1β, IL-6, IL-8, IL-10 and IL-12) - sometimes referred to as cytokine storm - were responsible for the induction of sepsis and rapid death. Inhibition of high-mobility group protein 1 (HMGB1) production in human macrophages and dendritic cells via siRNA treatment reduced lymphocyte apoptosis, decreased serum levels of HMGB1, TNF-α, IL20

1β, IL-6, IL-8, IL-10 and IL-12 and dramatically increased survival of the animals. This indicates that human patients with sepsis might benefit from neutralization of HMGB1 and that the humanized mouse model can be used not only to analyze the pathogenesis of sepsis but also test novel therapies.

Discussion

In humanized mice, the human cells and tissue allow for the infection with pathogens that have human tropism and the functional human immune system in this animal model enables scientists to study the responses against them. Research and testing of novel treatments such as drugs and vaccines can be performed in this animal model. Furthermore, human specific metabolism and side effects like liver toxicity can be investigated when the animals are engrafted with human hepatocytes [92,93]. However, humanized mice (especially the early animal models) also have limitations. One major limitation of the humanized mouse model is the species specificity of several hematopoietic growth factors. Murine IL-3 for example has no effect on human cells [94]. Since it is involved in survival, proliferation, and differentiation of multipotent hematopoietic progenitor cells and also plays an important role in the production of granulocytes, monocytes, erythrocytes and thrombocytes [95] it could be one of the reasons why these cell populations are underrepresented in humanized mice. Also, murine and human cytokines IL-15, GM-CSF, IL-4 and M-CSF, which play a role in the development of myeloid and NK cells, feature considerable differences in their sequences. Human cells also show little effect when exposed to the murine version of 21

these cytokines [96]. Various studies have shown that the injection or expression of human cytokines in humanized mice improves engraftment [96, 97]. Injection of IL-3 improves general engraftment in humanized mice [97] and expression of IL-15 increases the number of human NK cells [96]. Species-specific cytokines and growth factors not only play a role in the development of cells of the hematopoietic linage, but also control their differentiation and proliferation and, hence, influence the immune response. Expression of GM-CSF and IL-4 promote the maturation of B, T and dendritic cells thereby enhancing the humoral immune response [98]. Injection of recombinant IL-7 supports the development of T cells and therefore increasing their number in engrafted mice [14]. Another limiting factor of the humanized mouse model is the lack of adhesion molecules and other signaling proteins specific for human cells [12]. Adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1), are essential for proper leukocyte trafficking and also immune functions like binding of leukocytes to endothelial cells before transmigration into tissue. The importance of species-specific signaling proteins can be seen in the example of CD47 and its ligand SIRPα. SIRPα is expressed on phagocytic cells like macrophages. Binding of SIRPα to CD47 dampens phagocytic activity and reduces production of inflammatory cytokines such as TNF by phagocytes [99, 100]. Takenaka et al. showed that polymorphisms in SIRPα resulting in enhanced binding to human CD47 are responsible for the increased engraftment of human stem cells in mice with the non-obese diabetic (NOD) background [100]. Stem cell factor (SCF) is a surface protein and part of the microenvironment which supports the self-renewal capacity of HSC. While the ligand of human SCF (c-kit) does bind to mouse SCF, the binding 22

affinity is weaker. Takagi et al. were able to increase both the overall human CD45+ leukocyte levels and the number of human CD33+ myeloid cells by engrafting transgenic NSG mice that were expressing the membrane bound human SCF [101].

The adaptive immune response in humanized mice suffers from another limitation, which is the lack of HLA class I and II expression on mouse cells. The fact, that human T cells are positively selected in the thymus of humanized mice by murine epithelial cells featuring murine major histocompatibility complex (MHC) molecules impairs their function and also the function of B cells which require T cell support. The fact, that human MHC-restricted T cell responses as well as humoral immune responses and antibody class switches occur in humanized mice could be due to the positive selection of a limited number of T cells on human B and dendritic cells located in the murine thymus [41,102,103]. While B cells in humanized mice are capable of producing IgM and also lower levels of IgG, transgenic expression of human HLA class II is required for the class switching to and production of IgA and IgE. Human HLA class II expression also increases the number of T cells in humanized mice [102]. Humanized mice which express human HLA class I feature a higher number of cytotoxic T cells that recognize viral peptides in a HLA-restricted manner and exert HLA-restricted cytotoxicity compared to regular humanized mice [104]. These examples illustrate how administration of certain human cytokines or the expression of human cell surface molecules and proteins can improve existing humanized mouse models and thereby increase the usability of the animal model in translational research. The remaining question is which technique would be the most suitable one to 23

modify the human cytokine environment and the expression of surface molecules and proteins in humanized mice. The first option for genetically modifying the existing immunodeficient mouse strains, used for humanization, is the traditional embryonic stem cell technique. However, this method is time consuming and inefficient, and therefore currently superseded by new technologies in genetic engineering, like zinc-finger nucleases, transcription activatorlike effector nucleases, and CRISPR/Cas-based RNA-guided DNA endonucleases which make fast and efficient introduction of targeted modifications in mice possible [105]. These techniques can be used to knock-in genes of interest in mice [106-108] and, therefore, allow for the expression of human genes under the control of the murine promotor. Thus, not only the spacial but also the temporal expression will be physiological, which can be a critical requirement for example for growth factor expression during the development of the hematopoietic system. Another advantage of these methods is the fact, that once transgenic animals are created, they can be bred and therefore conveniently multiplied. However, despite new technologies, generating transgenic mice is still time consuming, requires special knowledge and is expensive. The second option for creating transgenic animals expressing selected human genes is backcrossing immunodeficient mouse strains with other mouse strains that already possess the human gene of interest. This technique was used to create humanized mice which express human HLA class I [104] and human HLA class II [102]. Creating transgenic mice using backcrossing with mice that already possess the desired mutation has the advantage of not requiring special knowledge and it is also a cost effective method. However, animals with the desired mutation and a suitable genetic background 24

have to be available for this method in order to be a feasible option. And the process is also time consuming. Another approach for expressing specific human genes in humanized mice can be achieved using viral vectors. O’Connel et al used a lentiviral vector to express human IL7 in humanized mice resulting in high serum levels of human IL-7 and an increase in human T cell numbers and lymphoid follicle size in the spleen [109]. Adenoviral vectors tend to be less useful for the expression of human genes in humanized mice, especially in animals serving as models for infectious diseases, because of their immunogenic property and the limited duration of transgene expression [110]. Retroviral vectors generally induce stable transgene expression, but can only transduce dividing cells. Due to their random insertion they can also cause oncogene activation or disrupt tumor suppressor genes. Lentiviruses are also retroviruses, but have the ability to infect both mitotic and post-mitotic cells. However, they still possess oncogenic potential. Using lentiviral vectors has the advantage of being fast and cost effective. The disadvantages are their oncogenic potential and a limited insert capacity of ~8 kb. Furthermore, the lentiviral transduction efficiency can vary widely, depending on the cell type that is transfected, and might, therefore, be unfavorable in certain cases. However, there are methods to increase transduction efficiency in certain cell types [111]. Another technology to express human genes in humanized mice is hydrodynamic tailvein injection, where hydrodynamic pressure is used to deliver genetic material into parenchyma cells [112]. This method has been successfully used to express human IL-15 and Flt-3/Flk-2 ligand in humanized mice resulting in increased serum levels of IL-15 and elevated NK cell levels [113]. Advantages of this method are that it is 25

straightforward and inexpensive. Similar to using retroviral vectors, hydrodynamic tailvein injection also allows the researcher to freely choose the point in time for expressing the desired gene in the animals without having to use a special promotor. The disadvantage of this system is the fact, that the target cells cannot be chosen freely, which makes this system only useful for expressing soluble factors like cytokines, and inept for the expression of surface proteins. Also, expression is only transient (expression of IL-15 in humanized mice only lasts 2-3 weeks [113]). The simplest technique for increasing the levels of human-specific soluble factors in humanized mice is direct injection of recombinant proteins. While this method is straightforward, easy to use, requires no special knowledge, and the point in time of the administration can be chosen freely, it also has drawbacks: It can only be used for soluble factors. Using recombinant proteins can also become expensive, especially when larger quantities of mice are needed for experiments. Furthermore, injection of soluble factors results in inconsistent plasma levels, being high directly after injection but declining until the next injection. This, however, can be mitigated by using a more sophisticated technique for administering soluble factors: the osmotic minipump. This device releases soluble factors like cytokines constantly resulting in a steady plasma level [114].

Conclusion

In conclusion it can be said, that the humanized mouse is a very valuable tool to study human-specific pathogens. This animal model might also be very useful to study the 26

pathogenesis of bacterial, viral and parasitic infections that can be studied in other in vivo models (e.g. immunocompetent mice), due to the fact that the immune response of the human immune system in these animals is probably better translatable to humans. Another very important aspect is the fact that the humanized mouse model has been used to successfully test not only approved but also new experimental drugs and to find novel treatment options as well. Currently only about 10% of new therapeutic agents manage to successfully complete the clinical trials and get approved as new molecular entities by e.g. the US Food and Drug Administration [115]. By using humanized mice, certain agents that show unforeseen off-target activity or low efficacy could be sorted out in preclinical testing. This would not only protect human study participants from adverse effects but also help in markedly reducing the cost of developing new therapeutic agents. However, the humanized mouse model still has certain constraints which have to be addressed in order to improve this animal model and also its predictive value when testing new drugs and vaccines.

Conflict of Interest No financial interest or any conflict of interest exists.

Acknowledgements

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36

Table 1: Viral infections Pathogen

Mouse strain

HIV

NOD/SCID/γc−/−

HIV

Rag2−/− γc−/−

Key findings

Ref

HIV-specific cellular and humoral immune response

31

Evolution and phenotypic changes of HIV in vivo due to

33

selective pressure by the immune system HIV

NOD/SCID/γc−/−

Intravaginal and rectal HIV infection possible

24,

Prophylaxis with anti-retroviral drugs prevents infection

25, 34

HIV

Rag2−/−γc−/− NOD/SCID/γc−/−

Treatment with novel transcription peptide (tat) inhibitor

36

reduces viral load

HIV

Rag2−/−γc−/−

Standard antiretroviral therapy reduces viral load

37

HIV

Rag2−/−γc−/−

Novel siRNA (tat/rev) treatment reduces viral load and CD4+

38

T cell depletion HIV

NOD/SCID/γc−/−

Novel shRNA (CCR5) reduces CCR5 expression and CCR5-

39

mediated HIV-1 infection HIV

Rag2−/−γc−/−

Human IL-7 expression increased T cell numbers but not viral

40

load EBV

NOD/SCID/γc−/−

Persistent infection, B cell lymphoproliferative disorder,

42

EBV-specific T cell response and IgM production EBV

NOD/SCID/γc−/−

HLA restricted and EBV specific cytotoxic T cell response

43

Infection induces EBV-associated hemophagocytic

44

Tg(HLA-A2.1) EBV

NOD/SCID/γc−/−

37

lymphohistiocytosis EBV

NOD/SCID/γc−/−

EBV

CB 17-SCID

EBV induces erosive arthritis resembling rheumatoid arthritis

45

Low dose IL-2 treatment prevents EBV-associated

46

lymphoproliferative disease EBV

CB 17-SCID

Rituximab and IL-2 have synergistic effect against B-cell

49

Non-Hodgekin’s Lymphoma HCV

HCV

Rag2−/−γc−/−

uPA-SCID

Infection induces hepatitis and fibrosis, HCV specific immune

52,

response

53

Direct-acting antiviral therapy selects therapy-resistant HCV

54

variants, combination of therapy with entry inhibitors prevents viral breakthrough HCV

uPA-SCID

Anti-scavenger receptor class B type I monoclonal antibody

55

therapy inhibits HCV infection HCV

FAH−/−Rag2−/− γc−/−

Expression of neutralizing antibodies via viral vectors protect

56

animals from HCV infection HBV

NOD/SCID/γc−/−

Persistent infection, chronic liver inflammation and fibrosis,

Tg(HLA-A2.1)

human immune response, HBV neutralizing antibody inhibits

57

infection and liver disease HCMV

NOD/SCID/γc−/−

HCMV establishes latent infection, G-CSF induces

58,6

reactivation of latent HCMV

0

HCMV

NOD/SCID/γc−/−

Novel HCMV locus mediates viral replication & latency

59

HCMV

SCID-Alb-uPA

Transferred human NK cells reduce levels of HCMV in

61

animals DENV

Rag2−/−γc−/−

Animals display plasma viremia and develop fever, DENV38

64

specific humoral immune response (IgM & IgG) DENV

DENV

NOD/SCID/γc−/−

NOD/SCID/γc−/−

Infection induces fever, viremia, erythema, and

65,6

thrombocytopenia similar to infected humans

6

Infection induced by mosquito bite leads to more severe

67

disease and innate & adaptive immune response DENV

NOD/SCID/γc−/− Tg(HLA-A2/Huβ2M)

DENV

NOD/SCID/γc−/−

Infection induces humoral (anti-DENV IgM) and HLA-

68

restricted T cell response Infection-induced thrombocytopenia caused by reduction of

69

megakaryocytes and progenitors DENV

NOD/SCID

Treatment with an adenosine nucleoside inhibitor decreases

70

viremia in infected animals HTLV-1

NOD/SCID/γc−/−

Infection induced adult T-cell leukemia in animals and

72

triggered HTLV-1-specific adaptive immune response HTLV-1

NOD/SCID/γc−/−

Treatment with HTLV-1-neutralizing antibodies prevents in

73

vivo transmission of virus HSV-2

Rag2−/−γc−/−

Vaginal infection induces protective innate and adaptive

74

immune response reducing local viral replication HuNoV

Rag−/−γc−/−

JCV

NOD/SCID/γc−/−

Successful infection and replication of HuNoV

75

Asymptomatic infection induced humoral and cellular

76

immune response against JCV Abbreviations: HIV, human immunodeficiency virus; NOD, non-obese diabetic; SCID, severe combined immunodeficiency; Rag, Recombination activating gene; tat, trans-activator of transcription, siRNA: small interfering RNA Rev: regulator of expression of virion proteins; CD, cluster of differentiation; CCR5, CC chemokine receptor type 5; IL, interleukin; EBV, Epstein-Barr virus; Ig, immunoglobulin; HLA, human leukocyte antigen; HCV, hepatitis C virus; uPA, urinary plasminogen activator; FAH, 39

Fumarylacetoacetase; tg, transgenic; HBV, hepatitis B virus; HCMV, human cytomegalovirus; G-CSF, granulocyte-colony stimulating factor; Alb, Albumin; NK, natural killer; DENV, Dengue virus; HTLV-1, human T cell leukemia virus type 1; HSV-2, herpes simplex virus type 2; HuNoV, human norovirus; JCV, John Cunningham virus

40

Table 2: Bacterial & parasitic infections Pathogen Plasmodium

Mouse strain NOD/SCID/γc−/−

Key findings

Ref

Improved disease model with higher infectious burden,

78

parasitemia decreases after therapy with proven and novel

falciparum

antimalaria drugs

Plasmodium

NOD/SCID

falciparum

NOD/SCID/γc−/−

Intravenous injection of erythrocytes and parasites results in

79

high parasite levels and long lasting parasitemia, different sexual stages of parasite present

Plasmodium

SCID Alb-uPA

uPA-SCID

Novel method for detection and quantification of parasites in

81

infected animals using qPCR

falciparum Leishmania

80

features infectious liver stages of parasite

falciparum Plasmodium

Improved model due to transplanted human hepatocytes

NOD/SCID/γc−/−

Adaptive and innate immune response against parasites,

82

therapy with Miltefosine partially effective, side effects of

major

treatment similar to human patients

Neisseria

SCID/Beige

Infection induces local vascular damage, thrombosis, vascular

84

leakage and purpura in skin grafts, type IV pili of pathogen

meningitides

identified as source for vascular dysfunction

Salmonella

Rag2−/−γc−/−

NOD/SCID/γc−/−

Immune response and pathological changes after infection,

86

previously unknown virulence determinants revealed

Typhi Salmonella

85

similar to human typhoid fever

Typhi Salmonella

Infection induces meningitis, liver pathology and mortality

Rag2−/−γc−/−

Persistent infection, human innate and adaptive immune 41

87

response with S. Typhi-specific antibodies

Typhi Salmonella

Rag2−/−γc−/−

Stable infection with high bacterial load in specific organs,

88

pathological changes including neurological symptoms, model

Typhi

for S. Typhi induced meningitis

Borrelia

NOD/SCID/γc−/−

89

fever, humoral immune response with pathogen-specific IgM

hermsii Streptococcus

Recurring bacteremia similar to Borellia-induced relapsing

NOD/SCID/γc−/−

Infection induced human cytokine production and leukocyte

90

trafficking, treatment leads to changes in leukocyte

agalactiae

populations and bacterial burden

intestinal

NOD/SCID/γc−/−

bacteria (CLP)

16

apoptosis which both exist in patients with sepsis

bacteria (CLP) intestinal

CLP induces human cytokine production and lymphocyte

NOD/SCID/γc−/−

CLP-induced sepsis leads to cytokine production and death, siRNA treatment reduces HMGB1 secretion, cytokine production, lymphocyte apoptosis and mortality

Abbreviations: NOD, non-obese diabetic; SCID, severe combined immunodeficiency; Rag, Recombination activating gene; Ig, immunoglobulin; uPA, urinary plasminogen activator; Alb, Albumin; qPCR, quantitative polymerase chain reaction; CLP, cecal ligation and puncture; HMGB1, high-mobility group protein B1

42

91