Discovery of a novel core inhibitor EP-027367 with potent antiviral activity both in vitro and in a humanized mouse model

ABSTRACT A series of 4-amino 2-anilinoquinazolines optimized for activity against the most lethal malaria parasite of humans, Plasmodium falciparum, was evaluated for activity against other human Plasmodium parasites and related apicomplexans that infect humans and animals. Four of the most promising compounds from the 4-amino 2-anilinoquinazoline series were equally as effective against the asexual blood stages of the zoonotic P. knowlesi, suggesting that they could also be effective against the closely related P. vivax, another important human pathogen. The 2-anilinoquinazoline compounds were also potent against an array of P. falciparum parasites resistant to clinically available antimalarial compounds, although slightly less so than against the drug-sensitive 3D7 parasite line. The apicomplexan parasites Toxoplasma gondii, Babesia bovis, and Cryptosporidium parvum were less sensitive to the 2-anilinoquinazoline series with a 50% effective concentration generally in the low micromolar range, suggesting that the yet to be discovered target of these compounds is absent or highly divergent in non-Plasmodium parasites. The 2-anilinoquinazoline compounds act as rapidly as chloroquine in vitro and when tested in rodents displayed a half-life that contributed to the compound’s capacity to clear P. falciparum blood stages in a humanized mouse model. At a dose of 50 mg/kg of body weight, adverse effects to the humanized mice were noted, and evaluation against a panel of experimental high-risk off targets indicated some potential off-target activity. Further optimization of the 2-anilinoquinazoline antimalarial class will concentrate on improving in vivo efficacy and addressing adverse risk.

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An EBNA3A-Mutated Epstein-Barr Virus Retains the Capacity for Lymphomagenesis in a Cord Blood-Humanized Mouse Model

Journal of Virology ◽

10.1128/jvi.02168-19 ◽

2020 ◽

Vol 94 (10) ◽

Author(s):

James C. Romero-Masters ◽

Makoto Ohashi ◽

Reza Djavadian ◽

Mark R. Eichelberg ◽

Mitchell Hayes ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

Mouse Model ◽

Cord Blood ◽

Wild Type ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) causes B cell lymphomas and transforms B cells in vitro. The EBV protein EBNA3A collaborates with EBNA3C to repress p16 expression and is required for efficient transformation in vitro. An EBNA3A deletion mutant EBV strain was recently reported to establish latency in humanized mice but not cause tumors. Here, we compare the phenotypes of an EBNA3A mutant EBV (Δ3A) and wild-type (WT) EBV in a cord blood-humanized (CBH) mouse model. The hypomorphic Δ3A mutant, in which a stop codon is inserted downstream from the first ATG and the open reading frame is disrupted by a 1-bp insertion, expresses very small amounts of EBNA3A using an alternative ATG at residue 15. Δ3A caused B cell lymphomas at rates similar to their induction by WT EBV but with delayed onset. Δ3A and WT tumors expressed equivalent levels of EBNA2 and p16, but Δ3A tumors in some cases had reduced LMP1. Like the WT EBV tumors, Δ3A lymphomas were oligoclonal/monoclonal, with typically one dominant IGHV gene being expressed. Transcriptome sequencing (RNA-seq) analysis revealed small but consistent gene expression differences involving multiple cellular genes in the WT EBV- versus Δ3A-infected tumors and increased expression of genes associated with T cells, suggesting increased T cell infiltration of tumors. Consistent with an impact of EBNA3A on immune function, we found that the expression of CLEC2D, a receptor that has previously been shown to influence responses of T and NK cells, was markedly diminished in cells infected with EBNA3A mutant virus. Together, these studies suggest that EBNA3A contributes to efficient EBV-induced lymphomagenesis in CBH mice. IMPORTANCE The EBV protein EBNA3A is expressed in latently infected B cells and is important for efficient EBV-induced transformation of B cells in vitro. In this study, we used a cord blood-humanized mouse model to compare the phenotypes of an EBNA3A hypomorph mutant virus (Δ3A) and wild-type EBV. The Δ3A virus caused lymphomas with delayed onset compared to the onset of those caused by WT EBV, although the tumors occurred at a similar rate. The WT EBV and EBNA3A mutant tumors expressed similar levels of the EBV protein EBNA2 and cellular protein p16, but in some cases, Δ3A tumors had less LMP1. Our analysis suggested that Δ3A-infected tumors have elevated T cell infiltrates and decreased expression of the CLEC2D receptor, which may point to potential novel roles of EBNA3A in T cell and NK cell responses to EBV-infected tumors.

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Blocking HIV-1 Infection by Chromosomal Integrative Expression of Human CD4 on the Surface of Lactobacillus acidophilus ATCC 4356

Journal of Virology ◽

10.1128/jvi.01830-18 ◽

2019 ◽

Vol 93 (8) ◽

Cited By ~ 3

Author(s):

Wenzhong Wei ◽

Joshua Wiggins ◽

Duoyi Hu ◽

Vladimir Vrbanac ◽

Dane Bowder ◽

...

Keyword(s):

Mouse Model ◽

Lactobacillus Acidophilus ◽

Sexual Transmission ◽

Effective Vaccine ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Antiviral Proteins ◽

Hiv 1

ABSTRACT Lactobacillus bacteria are potential delivery vehicles for biopharmaceutical molecules because they are well-recognized as safe microorganisms that naturally inhabit the human body. The goal of this study was to employ these lactobacilli to combat human immunodeficiency virus type 1 (HIV-1) infection and transmission. By using a chromosomal integration method, we engineered Lactobacillus acidophilus ATCC 4356 to display human CD4, the HIV-1 receptor, on the cell surface. Since human CD4 can bind to any infectious HIV-1 particles, the engineered lactobacilli can potentially capture HIV-1 of different subtypes and prevent infection. Our data demonstrate that the CD4-carrying bacteria are able to adsorb HIV-1 particles and reduce infection significantly in vitro and also block intrarectal HIV-1 infection in a humanized mouse model in preliminary tests in vivo. Our results support the potential of this approach to decrease the efficiency of HIV-1 sexual transmission. IMPORTANCE In the absence of an effective vaccine, alternative approaches to block HIV-1 infection and transmission with commensal bacteria expressing antiviral proteins are being considered. This report provides a proof-of-concept by using Lactobacillus bacteria stably expressing the HIV-1 receptor CD4 to capture and neutralize HIV-1 in vitro and in a humanized mouse model. The stable expression of antiviral proteins, such as CD4, following genomic integration of the corresponding genes into this Lactobacillus strain may contribute to the prevention of HIV-1 sexual transmission.

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In Vitro Expansion of Human Regulatory T Cells for Functional Study of Allograft Rejection in a Humanized Mouse Model

The Thoracic and Cardiovascular Surgeon ◽

10.1055/s-0036-1571713 ◽

2016 ◽

Vol 64 (S 01) ◽

Author(s):

L. Pauksch ◽

W. Sommer ◽

K. Jansson ◽

A.-K Knoefel ◽

T. Nakagiri ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Mouse Model ◽

Allograft Rejection ◽

Functional Study ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

In Vitro Expansion

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Pomalidomide Augments Erythropoiesis and Fetal Hemoglobin Production in a Humanized Mouse Model of Sickle Cell Disease

Blood ◽

10.1182/blood.v112.11.536.536 ◽

2008 ◽

Vol 112 (11) ◽

pp. 536-536 ◽

Cited By ~ 2

Author(s):

Steffen E. Meiler ◽

Marlene Wade ◽

Zhong Chen ◽

Preetha Ramalingam ◽

Laure A Moutouhde Parseval ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Spleen Weight ◽

Cell Disease ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Cd34 Cells

Abstract Introduction: Pomalidomide (PL) is an IMiD™ immunomodulatory compound. Thalidomide and lenalidomide have previously been shown to mitigate blood transfusion dependency in patients with thalassemia major and myelodysplastic syndromes, respectively.1,2 In a recent in vitro study, PL, which is currently being evaluated for the treatment of hematological cancers, stimulated erythropoiesis, F-cell production, total hemoglobin (Hb) and fetal hemoglobin (HbF) synthesis in human CD34+ cells.3 Furthermore, combined treatment of CD34+ cells with PL and Hydroxyurea (HU) synergistically upregulated HbF expression. These results suggest a promising role for PL in the treatment of β-hemoglobinopathies. To evaluate the in vivo efficacy of PL in sickle cell disease (SCD), we conducted an eight week study in a relevant knockout-transgenic (KT) mouse model. 4 Methods: Animals. Six week old KT homozygous sickle mice were treated daily (Mon–Fri; i.p. injections) for eight weeks with the following compounds: Grp 1. Vehicle (n=8); Grp 2. PL (10 mg/kg; n=9); Grp 3. HU (100 mg/kg; n=7); Grp 4. PL+ HU (n=8). Mice were maintained in an accredited pathogen-free animal facility according to NIH and institutional guidelines. Mice were anesthetized with Ketamine/Xylazine and blood collected by intracardiac puncture into 0.5 ml vacutainer EDTA tubes. Complete blood count (CBC) was analyzed with the CBC-Diff™ Veterinary Hematology System (Heska Inc., Loveland, CO). Reticulocyte counts were determined by supravital staining with methylene blue. HbF analysis of mouse hemolysates was done by HPLC using a weak cation-exchange column SynChropak CM-300 (Elchrom INC, Darien, IL). Organ analysis (liver, spleen, and femur marrow) included organ weights (%bw) and histology of H&E paraffin sections. Statistical analysis. One-Way ANOVA followed by Student-Newman-Kuels (Sigma Stat). Data are reported as the mean ± SE. A P-value of < 0.05 was considered significant. Results: Activity level and social behavior were unaffected by the treatment groups. There were no physical signs of drug toxicity and weight gains were comparable for all groups. PL significantly augmented HbF expression comparable to HU (HbF [peak%]: Veh: 6.24±0.35; PL: 9.51±0.83, P<0.01; HU: 10.54±0.77, P<0.01/total Hb [gm/dl]: Veh: 7.16±0.38; PL: 7.67±0.33; HU: 6.09±0.79). Surprisingly, PL-induced HbF production returned to control values after combinatory therapy with HU (HbF [peak%]: Veh: 6.24±0.35; PL+HU: 7.14±0.32). In contrast to HU, PL enhanced erythropoiesis as evidenced by increases in total RBCs, reticulocytes, and spleen weight (RBCs [×106/μl]: Veh: 4.84±0.14; PL: 5.49±0.19, P<0.05; HU: 4.47±0.51/Retics [%]: Veh: 36.81±5.78; PL: 40.47±4.73; HU: 16.41±3.94, P=0.01/Spleen weight [%bw]: Veh: 4.07±0.27; PL: 4.92±0.2, P=0.02; HU: 2.75±0.25, P<0.01). The total WBC count was largely unaffected by PL, but decreased significantly with HU (WBC [×103/μl]: Veh: 14.43±2.74; PL: 11.62±2.62; HU: 6.85±0.82, P<0.05). The PL group showed a trend toward increased marrow hyperplasia. A detailed analysis of cell density, erythroid and myeloid cells is underway. Liver histology revealed decreased tissue inflammation and focal necrosis in ~50% of PL-treated animals. Summary & Conclusions: 1. PL caused a robust induction of HbF in sickle mice. These results extend earlier in vitro findings of PL’s bioactivity in human erythroid progenitor cells. The HbF response to PL was similar to the established HbF-inducer HU, but surprisingly was lost in combination with HU. This inhibitory effect suggests that PL and HU stimulate HbF by distinct mechanisms. 2. Additionally, PL augmented erythropoiesis as demonstrated by increases in total RBCs, reticulocytes, spleen weight, and marrow hyperplasia. In contrast, HU showed the expected findings of generalized myelosuppression. 3. PL reduced liver injury and inflammation in ~50% of treated animals. Liver preservation tended to be associated with higher HbF values, though PL’s immunomodulatory properties may have contributed to this outcome. 4. In summary, PL exhibits a highly favorable hematological profile in a humanized mouse model of SCD. These results warrant further investigation in a Phase I trial of patients with SCD.

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A donor-dependent in vivo model for single agent and drug combination cytokine release syndrome safety evaluation.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.2612 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 2612-2612

Author(s):

James G. Keck ◽

Mingshan Cheng ◽

Michael Brehm ◽

Dale Greiner ◽

Lenny J. Shultz ◽

...

Keyword(s):

Mouse Model ◽

Single Agent ◽

Cytokine Release ◽

Cytokine Release Syndrome ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Human Pbmc ◽

Cytokine Levels

2612 Background: Although antibodies and CART cells therapies have been successfully used for cancer therapy, they can have lethal adverse effects such as cytokine release syndrome (CRS). The animal models and in vitro human PBMC assays presently in use can’t reliably predict the CRS in patients. A predictive marker for identifying patients at risk for developing CRS upfront would improve the safety of immune-oncology drug development. Methods: We have developed a rapid, sensitive and reproducible in vivo humanized mouse model for quantitating CRS. The NSG mouse and its derivatives are engrafted with human PBMCs. On day 6 we induced cytokines release with pembrolizumab, avelumab, atezolizumab, ipilimumab, anti-CD28, ATG and OKT3 in single dose; as well as combination treatments involving pembrolizumab, lenalidomide, ATG and anti-CD28. Furthermore, we compared our method versus the in vitro PBMC assay. The cytokine levels were also compared to the dose response. Results: There are about 10-15% CD45+ human cells on day 5 of engraftment; and among of them, there were approximately 70% CD3 T cells and 25% CD56 NK cells. All tested cytokines, human IFN-γ, IL-2, IL-4, IL-6, IL-10 and TNF were upregulated after 2 and 6 hours of OKT3, ATG, anti-CD28, pembrolizumab, avelumab and atezolizumab drug treatment. Mouse’s rectal temperatures dropped from 37-38 °C to about 36 °C at 6 hours’ time point in the treated groups. There is various cytokines release levels, low to high response in different donors with anti-CD28 treatment. All donors showed high response to OKT3. The cytokine release levels were consistent with a dose response or variable PBMC engraftment. The cytokine levels were also higher in some drug combination studies such as pembrolizumab combined with lenalidomide or ATG; anti-CD28 combined with ATG. Our in vivo method was able to determine CRS missed in the in vitro testing method. Conclusions: We have developed a rapid, sensitive and reproducible novel in vivo PBMC humanized mouse model that is able to differentiate human PBMC donors based on individual safety response to single agent and combination therapeutics of immune checkpoint inhibitors and possibly CAR-T therapy. This assay could be employed in future drug development.

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Functional Human CD141+ Dendritic Cells in Human Immune System Mice

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz432 ◽

2019 ◽

Vol 221 (2) ◽

pp. 201-213 ◽

Cited By ~ 3

Author(s):

Jordana G A Coelho-Dos-Reis ◽

Ryota Funakoshi ◽

Jing Huang ◽

Felipe Valença Pereira ◽

Sho Iketani ◽

...

Keyword(s):

Dendritic Cells ◽

Immune System ◽

Mouse Model ◽

Human Immune System ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Hematopoietic Stem ◽

Virus Serotype ◽

Human Immune

Abstract Background For the purpose of studying functional human dendritic cells (DCs) in a humanized mouse model that mimics the human immune system (HIS), a model referred to as HIS mice was established. Methods Human immune system mice were made by engrafting NOD/SCID/IL2Rgammanull (NSG) mice with human hematopoietic stem cells (HSCs) following the transduction of genes encoding human cytokines and human leukocyte antigen (HLA)-A2.1 by adeno-associated virus serotype 9 (AAV9) vectors. Results Our results indicate that human DC subsets, such as CD141+CD11c+ and CD1c+CD11c+ myeloid DCs, distribute throughout several organs in HIS mice including blood, bone marrow, spleen, and draining lymph nodes. The CD141+CD11c+ and CD1c+CD11c+ human DCs isolated from HIS mice immunized with adenoviruses expressing malaria/human immunodeficiency virus (HIV) epitopes were able to induce the proliferation of malaria/HIV epitopes-specific human CD8+ T cells in vitro. Upregulation of CD1c was also observed in human CD141+ DCs 1 day after immunization with the adenovirus-based vaccines. Conclusions Establishment of such a humanized mouse model that mounts functional human DCs enables preclinical assessment of the immunogenicity of human vaccines in vivo.

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