A CHIMERIC ANIMAL COMPRISING STABLY TRANSPLANTED BAT CELLS

Abstract

Generation of chimeric non-human animals hosting bat donor cells involves chimeric mice having bat cells that may be stably tolerated to provide a new platform technology in the general field of biology, and having application in the field of immunology related to virus-host interaction, cancer biology, autoimmunity, and the development of new drugs.

Claims

1-24. (canceled)

25. A chimeric non-human animal comprising xenogeneic transplanted bat cells.

26. The chimeric non-human animal of claim 25, wherein the bat cells give rise to at least one immune cell type selected from the group comprising monocytes, T cells, NK cells, B cells and dendritic cells.

27. The chimeric non-human animal of claim 25, wherein the non-human animal is a mouse acting as host to bat donor cells.

28. The chimeric non-human animal of claim 25, wherein the host is immunodeficient prior to receiving bat donor cells.

29. The chimeric non-human animal of claim 25, wherein the chimeric non-human animal does not develop graft versus host disease symptoms.

30. The chimeric non-human animal of claim 25, wherein the bat species is selected from the group comprising Eonycteris spelaean, Pteropus alecto and Myotis davidii.

31. The chimeric non-human animal of claim 27, wherein the mouse is NOD scid gamma (NSG) strain.

32. A method for producing the chimeric non-human animal of claim 25, comprising: a) providing bone marrow cells, splenocytes, stem cells or lymphoid cells from at least one bat; and b) introducing the bat bone marrow cells, splenocytes, stem cells or lymphoid cells into a neonatal or adult non-human animal which has been sub-lethally irradiated, wherein the non-human animal acts as host to bat donor cells.

33. The method of claim 32, wherein the host is a mouse.

34. The method of claim 32, wherein the host is immunodeficient prior to receiving the bat donor cells.

35. The method of claim 32, wherein the bat donor cells are introduced by intra-hepatic or intra-venous injection.

36. The method of claim 32, wherein the number of bat donor cells introduced is from 10.sup.3 cells.

37. The method of claim 32, wherein: a) the bat species is selected from the group comprising Eonycteris spelaean, Pteropus alecto and Myotis davidii, and/or b) the mouse is NOD scid gamma (NSG) strain.

38. The method of claim 32, further comprising collecting blood samples from the chimeric non-human animal and analysing for donor and host genes and/or peripheral blood type to confirm bat chimerism.

39. A method to study zoonotic viral infection, comprising; i) providing a plurality of the chimeric non-human animals of claim 25 and humanized mice, wherein the chimeric non-human animals are bat-NSG mouse chimeras; ii) infecting the mice of step i) with a zoonotic virus that causes disease in humans but does not cause clinical symptoms in host bats; iii) comparing the clinical symptoms and/or serological evidence of infection of the infected mice.

40. The method of claim 39, wherein the clinical symptoms and/or serological evidence of infection are selected from the group comprising fever, weight loss, antibody production, production of inflammatory cytokines, infection of bat immune cells, the presence of virus genetic material within infected mice and death.

41. The method according to claim 39, wherein the virus is Pteropine orthoreovirus (PRV), also known as Melaka virus.

42. The method of claim 39, wherein E. spelaean infected with Melaka virus is a negative control.

43. A method to study autoimmunity, comprising: i) providing a plurality of the chimeric non-human animals of claim 25 and humanized mice, wherein the chimeric non-human animals are bat-NSG mouse chimeras; ii) injecting a cohort of said bat-NSG mouse chimeras and NSG mice intra-peritoneally with an autoimmunity-inducing agent, regardless of sex and reconstitution level; iii) measuring the levels of human IgG, IgM, and anti-nuclear (anti-dsDNA, anti-histone, anti-RNP70, anti-SM and anti-SSA) IgGs in the plasma of the agent-injected bat-NSG mouse chimeras and NSG mice; and iv) comparing the levels for the injected bat-NSG mouse chimeras and NSG mice to the levels of bat IgG, IgM, and anti-nuclear (anti-dsDNA, anti-histone, anti-RNP70, anti-SM and anti-SSA) IgGs determined in the plasma of uninjected bat-NSG mouse chimeras and NSG mice.

44. The method of claim 43, wherein the autoimmunity-inducing agent is pristane [IUPAC name 2,6,10,14-tetramethylpentadecane].

45. The method of claim 43, wherein the bat-NSG mouse chimeras and NSG mice are injected at 12-13 weeks old.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0016] FIG. 1 shows measurement of reconstitution by qPCR and flow cytometry. Adult NSG mice were injected with 110.sup.6 bat bone marrow cells. (A) Detection of bat cells in bat-mice by species-specific primers. Peripheral blood samples were taken from NSG, C57BL/6, bat-mice 40-weeks post-injection, as well as bats, and assayed by qPCR with primers specific for mouse GAPDH SEQ ID Nos: 7-8, mouse 18S RNA SEQ ID Nos: 3-4, bat GAPDH SEQ ID Nos: 5-6 and bat 18S RNA SEQ ID Nos: 1-2. Data shown are the average cycle threshold (CT) values obtained from triplicates of each sample. (B-C) Forty-week post-injection, peripheral blood from NSG (B) (n=4) and bat-mice (C) (n=4) were stained for CD45.1, Ter119, CD11b, CD44, MHC-II and analyzed by flow cytometry. Concatenated staining profiles are shown. (D-E) Chimerism levels in peripheral blood of adult NSG mice, 10-weeks (D) and 40-weeks (E) after bat BM cell injection. Each symbol represents one mouse and the horizontal line indicates the mean value. (F) Proportions of various bat immune cell populations in a representative batch of bat-mice 40 weeks post-transplantation.

[0017] FIG. 2 shows assessment of potential GvHD in bat-mice. (A) Adult NSG mice were injected with 110.sup.6 bat spleen cells per mouse. Peripheral blood from bat-mice (n=4) were stained for CD45.1, Ter119, CD11b, CD44 and MHC-II and analyzed by flow cytometry. Concatenated staining profiles are shown. (B) Chimerism levels in peripheral blood of adult NSG mice injected with bat spleen cells, 40 weeks post-injection. Each symbol represents one mouse and the horizontal line indicates the mean value. (C) Proportions of various bat immune cell populations in a representative batch of bat-mice. (D) and (E) Histological analysis of organs from 40-weeks-old NSG control mice (n=4), NSG mice engrafted with 110.sup.6 C57BL/6 spleen cells 2 weeks post-injection (n=4), NSG mice engrafted with 110.sup.6 human PBMCs 4 weeks post-injection (n=4) and NSG mice engrafted with 110.sup.6 bat spleen cells 40 weeks post-injection (n=4). Paraffin slides made from indicated organs were processed and stained with H&E. Representative images are shown. Scale bar applies to all panels.

[0018] FIG. 3 shows in vivo transplantation of bat bone marrow (BM) cells with limiting dilution. Bat BM cells were isolated and injected into sub-lethally irradiated NSG pups at 510.sup.3 (5K), 133 10.sup.4 (10K), 510.sup.4 (50K) and 110.sup.5 (100K) cells per mouse (n=5 for each group). Forty weeks later, the mice were cheek-bled and blood was prepared and analyzed by flow cytometry. (A) Shown is the plot of chimerism levels within the peripheral blood of individual mice injected with indicated number of bat bone marrow cells. Each symbol represents one mouse and the horizontal line indicates the mean value. (B-D) Chimerism levels and total bat cell count in the (B) bone marrow, (C) liver and (D) spleen of individual mouse injected with indicated number of bat bone marrow cells 40 weeks post-transplantation. Each symbol represents one mouse and the horizontal line indicates the mean value. (E-H) Percentage proportion phenotypic lymphocyte subset analysis within the (E) peripheral blood, (F) bone marrow, (G) liver and (H) spleen of bat-mice injected with bat bone marrow cells.

[0019] FIG. 4 shows determination of antigen-specific antibody response in bat-mice. Bat BM cells were isolated and injected into sub-lethally irradiated NSG pups at 110.sup.5 cells per mouse (n=10). Twenty-four weeks post-injection, the mice were cheek-bled and blood was prepared and analyzed by flow cytometry. (A) Shown is the plot of chimerism levels within the peripheral blood of individual mice before challenge. Each symbol represents one mouse and the horizontal line indicates the mean value. (B) Bat-mice and NSG mice were challenged via intraperitoneal injection with NP-KLH, using IFA as an adjuvant. Plasma was collected from challenged bat-mice and NSG controls 2 weeks after the second booster. Optical density.sub.450 (OD.sub.450) levels of NP-specific antibodies were quantified by ELISA. Each symbol represents one mouse and the horizontal line indicates the mean value.

[0020] FIG. 5 shows measurement of reconstitution in organs from bat-mice by flow cytometry. Bat bone marrow (BM) cells were isolated and injected into sub-lethally irradiated NSG pups at 110.sup.5 (100K) cells per mouse (n=5). Forty weeks later, the mice were sacrificed; organs such as spleen, BM and liver were prepared, stained for CD45.1, Ter119, CD11b, CD44, and MHC-II and analyzed by flow cytometry. Concatenated staining profiles are shown.

[0021] FIG. 6 shows primers for qPCR. Primer sequences SEQ ID Nos: 1-8 for bat and mouse genes.

DETAILED DESCRIPTION OF THE INVENTION

[0022] Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.

Definitions

[0023] For convenience, certain terms employed in the specification, examples and appended claims are collected here.

[0024] As used herein, the term chimeric non-human animal refers to a non-human animal model composed of a mixture of cells from two different species. In preferred embodiments the non-human animal is a mouse which is then engrafted with cells from a bat.

[0025] As used herein, the term bat donor cells includes bone marrow cells, splenocytes, stem cells, lymphoid cells, cells from organs and/or tissues from bat origin.

[0026] As used herein, the term immunodeficient refers to severe defects in innate and adaptive immunity, a lack of immune cell compartments and deficiency in cytokine signaling.

[0027] As used herein, the term NSG is an abbreviation for NOD scid gamma and refers to the following mouse strain: NOD.Cg-PrkdC.sup.scid II2rg.sup.tm1Wjl/SzJ

[0028] As used herein, the term graft versus host disease (GVHD) is defined as a reaction that develops after an allogenic transplantation. GvHD is an immune-mediated disorder wherein the transplanted cells see the recipient's body as foreign, which results in the grafted cells attacking the new host.

[0029] As used herein, the term comprising does not preclude the presence of additional steps or substances in the methods and non-human chimeric animals, respectively, of the invention, and is understood to include within its scope the terms consisting of and consisting essentially of features defined in the claimed invention.

[0030] The inventors have found that bat cells, such as bat bone marrow cells, splenocytes, stem cells and/or lymphoid cells can be transferred into a different species of non-human animal to generate a chimeric animal having a functional bat immune system and no apparent bat cell/tissue rejection.

[0031] Accordingly, in a first aspect, the present invention provides a chimeric non-human animal comprising xenogeneic transplanted bat cells. It would be understood that the non-human animal is not a bat and receives donor cells from a bat to become chimeric.

[0032] In a preferred embodiment of the invention, the bat cells are selected from a group comprising bone marrow cells, splenocytes, stem cells and lymphoid cells.

[0033] In another preferred embodiment of the invention, the bat cells give rise to at least one immune cell type selected from the group comprising monocytes, T cells, NK cells, B cells and dendritic cells.

[0034] In another preferred embodiment of the invention, the non-human animal is a mouse acting as host to bat donor cells.

[0035] In another preferred embodiment of the invention, the host is immunodeficient prior to receiving bat donor cells.

[0036] In another preferred embodiment of the invention, the chimeric non-human animal does not develop graft versus host disease symptoms.

[0037] In another preferred embodiment of the invention, the bat species is Eonycteris spelaea.

[0038] In another preferred embodiment of the invention, the bat species is Pteropus alecto.

[0039] In another preferred embodiment of the invention, the mouse is NOD scid gamma strain. It would be understood that other strains of immunodeficient mice would be suitable as host to the bat cells.

[0040] In a second aspect, the present invention provides a chimeric non-human animal according to any aspect of the invention for use as a model to develop bat antibodies, to study infectious diseases, autoimmunity, aging, cancer or graft versus host disease.

[0041] In a third aspect, the present invention provides a method for producing a chimeric non-human animal comprising bat cells, comprising the steps; [0042] a) providing bone marrow cells, splenocytes, stem cells or lymphoid cells from at least one bat; and [0043] b) introducing the bat bone marrow cells, splenocytes, stem cells or lymphoid cells into a non-human animal which has been sub-lethally irradiated,
wherein the non-human animal acts as host to bat donor cells.

[0044] In a preferred embodiment of the third aspect of the invention, the non-human animal is a neonate or adult.

[0045] In a preferred embodiment, the host is a mouse.

[0046] It is understood that the optimum time to create humanized mouse models with the highest level of engraftment appears to be by injecting neonatal mice within about 48 hours of birth. However the inventors have found that successful engraftment of bat cells into a mouse may also occur as late as at about 8 weeks of age.

[0047] In another preferred embodiment of the invention, the host at the time of engraftment is an 8-week-old adult mouse.

[0048] In another preferred embodiment of the invention, the host is a neonatal mouse engrafted within 48 hours of birth.

[0049] In another preferred embodiment of the invention, the host is immunodeficient prior to receiving the bat donor cells.

[0050] In another preferred embodiment of the invention, in step b) the bat donor cells are introduced by intra-hepatic or intra-venous injection.

[0051] In another preferred embodiment of the invention, the number of bat donor cells introduced is from 110.sup.3 cells; such as from 510.sup.3, from 110.sup.4, from 510.sup.4, from 110.sup.5, from 510.sup.5, from 110.sup.6, from 510.sup.6 cells, from 110.sup.7 cells or any suitable number. Preferably at least 110.sup.5, more preferably at least 110.sup.6, bat donor cells are introduced.

[0052] Although the bat species exemplified herein is Eonycteris spelaean, other bat species may be suitable as cell donors for producing a chimeric non-human animal comprising bat cells.

[0053] In another preferred embodiment of the method of the invention, the bat species is selected from the group comprising Eonycteris spelaean, Pteropus alecto and Myotis davidii.

[0054] In another preferred embodiment of the method of the invention, the mouse is NOD scid gamma strain.

[0055] In another preferred embodiment, the method further comprises collecting blood samples from the chimeric non-human animal and analysing for donor and host genes and/or peripheral blood type to confirm bat chimerism.

[0056] In a fourth aspect, the present invention provides use of a chimeric non-human animal according to any aspect of the present invention, for developing bat antibodies, to study infectious diseases, autoimmunity, aging, cancer or graft versus host disease.

[0057] In a preferred embodiment of the invention there is provided a method to study zoonotic viral infection, comprising;

[0058] i) providing NSG bat-mice and humanized mice;

[0059] ii) infecting the mice of step i) with a zoonotic virus that causes disease in humans but does not cause clinical symptoms in host bats;

[0060] iii) comparing the clinical symptoms and/or serological evidence of infection of the infected mice.

[0061] In a preferred embodiment, the clinical symptoms and/or serological evidence of infection are selected from the group comprising fever, weight loss, antibody production, production of inflammatory cytokines, infection of bat immune cells, the presence of virus genetic material within infected mice and death.

[0062] In a preferred embodiment, the virus is Pteropine orthoreovirus (PRV), also known as Melaka virus, documented to cause fever and acute respiratory diseases in human.

[0063] In a preferred embodiment, E. spelaea infected with Melaka virus is a negative control.

[0064] A bat-mouse model that does not display any clinical symptoms and/or serological evidence for Melaka infection, would be a good model to study other zoonotic virus infection. A screen for suitable infectious agent can be performed, to determine the suitable viruses and the optimum multiplicity of infection (MOI) ratio for each virus. How the bat immune system avoids clinical symptoms can also be investigated.

[0065] In a preferred embodiment of the invention there is provided a method to study autoimmunity, comprising;

[0066] i) providing NSG bat-mice and NSG mice;

[0067] ii) injecting a cohort of said NSG bat-mice and NSG mice intra-peritoneally with an autoimmunity-inducing agent, regardless of sex and reconstitution level;

[0068] iii) measuring the levels of human IgG, IgM, and anti-nuclear (anti-dsDNA, anti-histone, anti-RNP70, anti-SM and anti-SSA) IgGs in the plasma of the autoimmunity-inducing agent-injected bat-mice and NSG mice; and

[0069] iv) comparing the levels for the injected bat-mice and NSG mice to the levels of bat IgG, IgM, and anti-nuclear (anti-dsDNA, anti-histone, anti-RNP70, anti-SM and anti-SSA) IgGs determined in the plasma of uninjected bat-mice and NSG mice.

[0070] In a preferred embodiment, the autoimmunity-inducing agent is pristane [IUPAC name 2,6,10,14-tetramethylpentadecane].

[0071] In a preferred embodiment, the NSG bat-mice and NSG mice are injected at 12-13 weeks old.

[0072] In a preferred embodiment, the levels are measured using ELISA quantification.

[0073] In another preferred embodiment of the method of the invention, the bat species is selected from the group comprising Eonycteris spelaean, Pteropus alecto and Myotis davidii.

[0074] Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES

[0075] Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).

[0076] In this study, we adopted the concept of humanized mouse models and aimed to stably reproduce bat's biological system, particularly the immune system, in mice, by transplanting bat cells (Eonycteris spelaea) into immunodeficient mouse strain NOD-scid IL-2R.sup.-/- (NSG) mice (to create what we describe as bat-mice). In this bat-mouse model, bat immune cells were successfully reconstituted in mice and unique features, such as resistance to GvHD and functional demonstration of antigen-specific bat antibody responses were observed and characterized.

Example 1

Materials and Methods

Mice

[0077] NOD-scid IL-2R.sup.-/- (NSG) mice were obtained from Jackson Laboratory and bred in the animal facility at A*STAR, Biological Resource Centre in Singapore. Neonatal mice within 48 hours of birth (100 rads), and 8-week old adult mice (250 rads) were sub-lethally irradiated and injected with bat cells via intra-hepatic and intravenous injection, respectively.

Bat Cell Isolation from Eonycteris spelaea

[0078] Eonycteris spelaea (common name, cave nectar bat) were captured in Singapore at dusk using mist nets and transferred to clean customized bat bags for transportation. All animal processing work was conducted in accordance with approved guidelines, methods and permits from Duke-NUS Medical School and SingHealth Experimental Medicine Centre (2015/SHS/1088). Bats were anaesthetised using isoflurane and exsanguinated via cardiac bleed. The bat was sprayed with alcohol to decontaminate the fur coat, followed by an incision from the chest to the pubis area. The spleen tissues were mashed through a 100 m filter (Thermo scientific, USA) in DMEM medium (Thermo scientific, USA). The single-cell suspension was washed and re-suspended in media supplemented with 10% fetal bovine serum (FBS). Bone marrows were processed by making incisions on both ends of the femurs and tibias and flushing them with 5 ml of DMEM medium using 5 ml syringes (BD Biosciences, USA) and 27 gauge needles (BD Biosciences, USA). Contents flushed out of the bone marrow were mashed through a 100 m filter in DMEM medium. The single-cell suspension was washed and re-suspended in 5 ml red blood cell lysis buffer and incubated at room temperature for 5 minutes. The cell suspension was washed and resuspended with media supplemented with 10% FBS. Cell viability was assessed using trypan blue. Prior to delivery of cells to NSG mice, cells are washed and resuspended in StemSpan Serum-Free Expansion Medium.

Bat Cell Isolation from Pteropus alecto

[0079] The bat species Pteropus alecto, is obtained from the Australia Bat Clinic and Wildlife Trauma Centre. Splenocytes and bone marrow cells are isolated using generally the same method as above. For these bats bone marrow cells are also harvested from the ribs and a 23 gauge needle filled with complete media is used to flush out the cells. The bone marrow cell suspension is also treated and processed for the purpose of reconstituting a bat-mouse as described for E. spelaea.

Processing of Mouse/Bat-Mouse Tissue

[0080] For processing of bat-mouse tissues, organs are aseptically removed from the sacrificed bat-mouse. Spleen, liver, lymph node and kidney are cut into smaller pieces and mashed through the 100 m sieve to create single cell suspensions of the respective organs. For bone marrow cells, incisions are made on each end of the mouse tibia and femur and cells are flushed out using an 18 gauge needle and complete media. Cell suspensions are centrifuged at 1200 rpm at 4 C. for 5 minutes and supernatants are discarded. The cell pellets are resuspended in 5 ml red blood cell lysis buffer and incubated at room temperature for 5 minutes. After incubation, 7 ml of complete media is added to individual tubes. Tubes are spun at 1200 rpm at 4 C. for 5 minutes. Washing step is repeated once. Supernatants are discarded and cell pellets are resuspended in complete media or flow cytometry labelling cells (FACS) buffer.

Flow Cytometry

[0081] Conjugated antibodies for mouse CD11b (MI/70; BD Biosciences, USA), MHC-II (2G9; BD Biosciences, USA), CD45.1 (A20; Biolegend, USA), Ter119 (TER-119; Biolegend, USA) and CD44 (IML; eBioscience, USA) were used in flow cytometry assays. Cells were stained with antibodies in 100 l PBS containing 0.2% BSA and 0.05% sodium azide for 30 minutes on ice. Flow cytometry was performed on an LSRII flow cytometer using the FACSDiva software (BD, USA); 110.sup.4 events were collected per sample, and analyzed using the Flowjo software version 10 (Treestar, Ashland, USA). Percentage chimerism was calculated using the formula, [% mCD45.1.sup.Ter119.sup./(mCD45.1 .sup.Ter119.sup.+mCD45.1.sup.+Ter119.sup.)].

Genomic DNA Extraction from Bat-Mouse

[0082] DNA extraction is performed according to the method described in E.Z.N.A Tissue DNA Kit, product number: D3396-02.

[0083] However, instead of mincing the tissue, tissues are homogenized via FastPrep-24 5G Sample Preparation System (MP Biomedicals). 30 mg of tissues are added into a 2 ml Eppendorf tube with 500 g of silicon carbide, 400 l TL buffer and 25 l of OB protease solution. Both TL buffer and OB protease solution are included in the E.Z.N.A Tissue DNA Kit. Samples are homogenized by FastPrep-24 5G Sample Preparation System (MP Biomedicals) at 6.0 m/sec for 1 minute. After homogenization, samples are spun at 10,000g for 5 minutes. Supernatants are collected and transferred into new Eppendorf tubes.

RNA Isolation and Quantitative Polymerase Chain Reaction (qPCR)

[0084] Blood samples were collected from NSG, C57BL/6, bat-mice and bats. RNA was prepared from these samples using RNeasy Micro kit (Qiagen, Netherlands). Reverse transcription was performed using iScript cDNA Synthesis Kit (BIO-RAD, USA) according to manufacturer's specifications. qPCR was subsequently performed in triplicates using SensiFAST SYBR No-ROX Kit (Bioline, USA) and assays were run on the CFX96 Touch Real-Time PCR Detection System (BIO-RAD, USA) under the following cycling condition: 95 C. for 5 minutes, followed by 40 cycles of 95 C. for 5 seconds and 58 C. for 30 seconds, and ending with a melt profile analysis. Sequences of bat and mouse specific primers are listed in FIG. 6 and sequence listing as SEQ ID NOs: 1-8.

Mouse Immune Challenge

[0085] Mice were challenged by injecting NP-KLH (Biosearch Technologies, USA) precipitated to IFA (Sigma-Aldrich, USA), intraperitoneally. To prepare precipitation, NP-KLH and IFA were added together and sonicated. Three injections of 100 g of NP-KLH were used to challenge the mice. After the first challenge, mice were supplemented with booster shots which were injected on the same day for 2 consecutive weeks. Mice were bled before challenge and bled again 2 weeks following the third injection for the detection of NP-specific antibodies.

ELISA Detection of Bat Ig in Immune-Challenged Mice

[0086] Plasma was collected before and after challenging with NP-KLH by cheek bleeds or tail bleeds. Bat immunoglobulin (Ig) content specific to NP-KLH antigens was assessed by ELISA. Briefly, microtiter plates (Thermo Fisher Scientific, USA) were coated with NP31-BSA (Biosearch Technologies, USA) at 10 g/ml. Due to the lack of anti-bat secondary antibody, pierce recombinant protein A/G horseradish peroxidase conjugate (Thermo Fisher Scientific, USA) was used as a detection reagent to Ig in plasma. As NSG mice do not have mouse T and B cells, which results in an absence of mouse Ig, the Ig detected in bat-mice sera is of bat origin. Optical density (OD) was read at 450 nm with a plate reader (Tecan, USA).

Histology

[0087] Mouse organs were collected, fixed with 10% formalin and embedded in paraffin for processing into sections. Formalin-fixed paraffin sections (4-6 m) were dewaxed by melting for 30 minutes at 65 C., cleared in xylene twice for 5 minutes, and rehydrated in water-ethanol solutions containing decreasing percentages of ethanol. To determine tissue morphology, sections were stained with hematoxylin-eosin (Gill 2 Hematoxylin and Eosin Y alcoholic; Thermo Sandon, Cheshire, UK) following a standard procedure. Sections were imaged and analysed under an Olympus BX-61 microscope (Olympus, Japan).

Statistical Analysis

[0088] Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software Inc). Pairwise comparison was performed using two-tailed t test. P value less than 0.05 is considered statistically significant. All data are represented as mean.

Example 2

Transplantation of Bat Bone Marrow (BM) Cells Led to Stable Reconstitution of Bat Immune Cells in NSG Recipients

[0089] Due to their immunodeficiency, NSG mice are permissive for the engraftment of foreign cells. To investigate their ability to support the engraftment of bat cells, 8 week old adult NSG mice were exposed to 2.5 Gy (250 rads) irradiation in an X ray irradiator. Post-irradiation, 110.sup.8 whole bat BM cells were transferred intravenously into the treated mice. Ten weeks post-transplantation, blood samples were collected and analyzed for bat and mouse genes with species-specific primers SEQ ID Nos: 1-8 (FIG. 6) by quantitative polymerase chain reaction (qPCR). Untreated NSG mice were also kept, in parallel, as controls. Wild bat and C57BL/6 mice were used as both positive and negative controls, depending on the species-specific primers used. The results showed the presence of bat housekeeping genes (GAPDH; SEQ ID Nos: 5-6 and 18S; SEQ ID Nos: 1-2) within bat-mice, but not in control NSG mice (FIG. 1A). To further dissect the immune cell composition in blood, peripheral blood from both NSG and bat-mice were analyzed via flow cytometry. Due to the lack of bat-specific antibodies, antibodies such as anti-mouse CD11b, CD44 and major histocompatibility complex class II (MHC-II), which showed cross-reactivity with bats previously and are able to specifically bind to E. spelaea cells, were used. As shown in FIG. 1B and 1C, mouse-specific CD45.1 and Ter119 antibodies were used to gate out the majority of the mouse leukocytes and erythroid lineage cells. CD45.1.sup.Ter119.sup. population was further separated into two populations by CD44 and CD11b antibodies. Within the CD44.sup.+CD11b.sup.+ population, monocytes and dendritic cells (DCs) were gated out by further staining with CD44 and MHC-II. From CD44.sup.+CD11b.sup. cell population, CD44 and MHC-II were used to distinguish between T/natural killer (NK) and B cells. All of the major bat immune cell populations, such as monocytes, T/NK cells, B cells and DCs were found within the peripheral blood of bat-mice (FIG. 10). Bat chimerism in bat-mouse peripheral blood was calculated by analyzing the cells that stained negative for both mouse CD45.1 and Ter119, using the formula: [% mCD45.1.sup.Ter119.sup./(mCD45.1.sup.Ter119.sup.+mCD45.1.sup.+Ter119.sup.)]. At 10 weeks post-reconstitution, the percentage of bat chimerism ranged from 7% to 9% in the peripheral blood of four separate bat-mice in the group (FIG. 1D). Forty weeks post-transplantation, an increase in reconstitution levels, ranging from 20% to 40%, was observed, proving long-term reconstitution is viable in the bat-mouse model (FIG. 1E). Based on the profile of bat immune cells, monocytes made up the largest proportion of cells at 45% to 50%, followed by B cells at 20% to 25%, T/NK cells at 30% to 32% and DCs at 0.2%-1%. Bat immune cell profiles from a representative batch of bat-mice is shown in FIG. 1F. The low proportion of DCs could not be visualized due to the graphical scale used in FIG. 1F. Taken together, these results indicate that bat BM cells have a long-term repopulating capacity to survive, expand and stably establish a bat immune system in NSG recipients.

Example 3

Generation of P. alecto Bat-Mouse

[0090] Pregnant NSG mice are closely monitored towards the end of their term. 24-72-hour old pups are placed in sterile petri dishes and transferred to a sterile filter secondary container to be transported to the X ray irradiator. The pups are exposed to a total of 1 Gy (100 rads) radiation. After irradiation, pups are injected with 1-510.sup.5 bone marrow cells or splenocytes from Pteropus alecto via the intrahepatic route. A 30 G insulin needle is used for intrahepatic injection due to the small volume injected. No anaesthesia is used on the pups as anaesthesia poses a higher death risk to the pup. After injection of bat cells, the pups are placed back into the petri dish and the sterile secondary container to be transported back to the vivarium. Pups are monitored closely for the next 72 hours. Any pup that displays any stress symptoms, is euthanized via placement on ice for 10 minutes. Reconstituted pups are weaned from their mother between 4-5 weeks old. Cheek bleeds are performed at 10 weeks post-reconstitution.

Example 4

Bat Cells Did Not Induce Graft Versus Host Disease (GvHD) in NSG Mice

[0091] Transplantation of an autologous or syngeneic graft will not trigger a rejection. However, with an allogeneic graft, wherein the donor and recipient are genetically different, the recipient will develop GvHD. The transfer of mature bat lymphoid cells was expected to cause the development of GvHD symptoms. However, it was surprising to observe that the transplantation of bat cells did not lead to any signs of GvHD in bat-mice, even 40 weeks after initial cell injection. To investigate if bat cells would generate a rejection response in NSG mice, bat splenocytes (110.sup.6) were used for transplantation, as the majority of the cells within this organ are mature immune cells [Reddy, P., Negrin, R. & Hill, G. R. Biol. Blood Marrow Transplant. 14: 129-135 (2008)]. Forty weeks after injection, mice appeared healthy with no signs of GvHD being observed. Monocytes, T/NK cells, B cells and DCs were present in NSG mice engrafted with bat splenocytes (FIG. 2A). The reconstitution levels of bat immune cells in these mice ranged from 12% to 15% (FIG. 2B). The overall proportions of bat monocytes, T/NK, B cells and DCs were 60% to 65%, 30% to 38%, 5% to 10% and 0-1%, respectively, in peripheral blood. Bat immune cell profiles in peripheral blood from a representative batch of bat-mice is shown in FIG. 2C. The low proportion of DCs could not be visualized due to the graphical scale used in FIG. 2C. The successful reconstitution with bat splenocytes suggested that bat mature immune cells might have the ability to expand and subsequently lead to long-term in vivo repopulation. This novel finding has never been observed before in species such as humans, mice and other mammals.

[0092] In order to uncover if bat-mice had immune-mediated tissue damage from GvHD in the absence of observable clinical signs, histological analysis was applied to assess pathological changes in different organs from bat-mice. In most acute symptoms of GvHD, the first organs affected with tissue damage are the liver, skin and intestinal tract. Transplantation of C57BL/6 splenocytes and human peripheral mononuclear cells (PBMCs) separately into NSG mice was used as a positive control as they have been known to induce GvHD in recipients with different genetic backgrounds. Two to four weeks post-transplantation, recipients of 110.sup.6 C57BL/6 splenocytes or 110.sup.6 human PBMCs displayed symptoms of runt disease, a condition that features small and weakened mice, characteristic of GvHD [Korngold, R. & Sprent, J. J. Exp. Med. 148: 1687-1698 (1978)]. Organs were harvested from these mice and compared to bat-mice by histological and pathological analysis. Massive cell infiltration and damage were observed in various organs from mice that received C57BL/6 splenocytes or human PBMCs, while there was no significant difference between NSG mice and bat-mice, both of which were without any evident signs of GvHD (FIG. 2D and 2E). These results confirmed that mature bat immune cells did not induce GvHD in NSG mice. In addition to the successful reconstitution of bat immune cells in mice, the present invention reveals this important and unexpected outcome: that successful engraftment of mature immune cell xenograft can be achieved with no signs of rejection. This new model will serve as a useful tool to explore deeper mechanisms on how bat immune cells develop resistance to GvHD while maintaining their functionality. Such research will also aid in the development of drugs for use in curtailing rejections in transplantations.

Example 5

Optimization of Bat Cell Engraftment in NSG Mice

[0093] To optimize the production of bat-mice, further studies were conducted to improve the transplantation protocol and define the minimum number of bat BM cells needed to reconstitute NSG mice [Yong, K. S. M. et al. Cell. Mol. lmmunol. 13: 605-614 (2016); Ploemacher, R. E., van der Sluijs, J. P., Voerman, J. S. & Brons, N. H. Blood 74: 2755-2763 (1989); Taswell, C. J. Immunol. 126: 1614-1619 (1981)]. As compared to adult mice, neonatal NSG are known to be better at establishing human cell repopulation in humanized mice [Drake, A. C., Chen, Q. & Chen, J. Cell. Mol. Immunol. 9: 215-224 (2012)]. Bat BM cells at varying numbers of 510.sup.3, 110.sup.4, 510.sup.4 or 110.sup.5 cells were prepared and injected into sub-lethally irradiated NSG neonates within 48 hours of birth. As shown in FIG. 3A, according to peripheral blood reconstitution levels at 40 weeks post-injection, the groups of 510.sup.3 and 110.sup.4 cells did not give rise to significant reconstitution, while a transfer of 510.sup.4 or more cells resulted in engraftment. However, to achieve a robust establishment and maintenance of bat immune cells in NSG recipients, an initiating number of 110.sup.5 bat BM cells was required to reach a reconstitution level of -20% to 50% (FIG. 3A), which was comparable to adult NSG mice receiving 110.sup.6 BM cells (FIG. 1E). Systemic reconstitution in various organs such as BM, liver and spleen was analyzed (FIG. 5). The bat cell chimerism levels in organs from groups of 510.sup.3, 110.sup.4 and 510.sup.4 cells remained low. In mice injected with 110.sup.5 cells, the reconstitution levels were robust, with variation in different organs: in BM, the level of bat chimerism was _20% to 35%, with bat cell numbers ranging from 210.sup.6 to 310.sup.6 (FIG. 3B); in liver, reconstitution levels could reach 50% to 75%, with bat cell numbers of 110.sup.6 to 210.sup.6 (FIG. 3C); spleen had the highest reconstitution levels and cell numbers, which were 70% to 80% and 2010.sup.6 to 4010.sup.6, respectively (FIG. 3D). The dramatic increase in the number of bat cells within the organs, compared to the initiating number of cells injected, demonstrated that there was a massive in vivo expansion of bat cells in NSG mice (FIG. 3B to 3D). Overall proportions of immune subsets, such as monocytes, T/NK, B cells and DCs also varied between the peripheral blood, bone marrow, liver and spleen. In the peripheral blood, monocyte levels were 40% to 70%, T/NK cells 20% to 58%, B cells 2% to 5% and DCs 2% to 5% (FIG. 3E). In the bone marrow, monocytes 80% to 95%, T/NK cells 2% to 5%, B cells 2% to 5% and DCs 2% to 5% (FIG. 3F). Within the liver, the proportion of monocytes was 60% to 62%, T/NK cells 20% to 30%, B cells 10% to 20% and DCs 0% to 0.05% (FIG. 3G). The spleen was mostly dominated by monocytes, 90% to 95%, with T/NK, B cells and DCs standing at 2% to 5%, 1% to 3% and 0.5% to 2%, respectively (FIG. 3H). The low proportion of DCs in FIGS. 3E-H could not be visualized due to the graphical scale used.

[0094] Altogether, it is evident that neonatal mice, with an engraftment of a limited number of bat BM cells, were able to achieve considerable levels of chimerism with all major immune cells present, thereby enabling the reliable generation of a large cohort of bat-mice. Using the optimised protocol, we envisage that 80 to 100 bat-mice could potentially be generated from the BM of a single E. spelaea bat.

[0095] The unexpected discovery that bat BM cells can be successfully engrafted without rejection or GvHD is highly significant on two accounts. First, it may represent one of the strongest pieces of evidence obtained to date in support of the notion that there is immune tolerance or a dampened innate defence system in bats. Secondly, this phenomenon, which is not typically seen in mainstream immunological studies of other mammals, may open a new area for the study of immunity in unique species and create new opportunities to address human health issues. Together, this would not only allow a better understanding of bat immune responses to diseases and vaccines, but also provide a means for testing immune-modulators and exploring mechanisms in infection and neoplasia.

Example 6

The Reconstituted Bat Immune System is Functional in Bat-Mice

[0096] The demonstration of a functional bat immune system in NSG mice recipients holds a fundamental importance for the potential utility of this model. To investigate whether an antigen-specific adaptive immune response could be achieved in bat-mice, 24-week-old bat-mice that were generated by injecting 110.sup.5 BM cells during their neonatal stage (FIG. 4A), were challenged with 4-Hydroxy-3-Nitrophenylacetyl hapten conjugated to Keyhole Limpet Hemocyanin (NP-KLH) using Incomplete Freund's Adjuvant (IFA) as an adjuvant. An ELISA-based system was customized to detect NP-specific bat antibodies using NP31-BSA as the ELISA antigen so that only NP-reacting antibodies would lead to a positive reading. Plasma samples from the 10 challenged bat-mice all showed positive reactivity with NP-antigen, whereas the pre-bleed of each bat-mouse and samples from the challenged NSG control mice were all negative (FIG. 4B). These results confirmed that bat-mice were able to generate an antigen-specific response, thereby indicating the presence of functional immune cells.

[0097] The observation of a rapid development of bat-specific antibodies post-NP-KLH challenge suggests that bat humoral immune responses involving antigen presenting cells and B cells within a mouse environment are intact and functional. Additionally, as KLH is known to be a T cell-dependent antigen which primes antigen-specific T cell responses, the responses to NP-KLH suggests that bat-mice could have developed antigen-specific T cell responses.

Example 7

Viral Infection

[0098] To investigate the feasibility of using the bat-mouse model of the present invention for viral infection studies, both E. spelaea and P. alecto bat-mice are generated as described above. In addition, humanized mice are generated to be used as a positive control. A suitable candidate virus is Pteropine orthoreovirus (PRV), also known as Melaka virus. Bats have been described to be the natural host for Melaka virus and do not display any clinical symptoms upon infection. By contrast, Melaka virus has been documented to cause fever and acute respiratory diseases in human. Hence, the humanized mice will display similar symptoms, such as fever and weight loss and show serological evidence of infection such as antibody production, production of inflammatory cytokines and presence of Melaka virus genetic material within infected humanized mice. Some may even die as a result of the infection. By comparison, the bat-mice do not display any clinical symptoms as the bat's immune system is immune-dampened upon virus infection. Clinical symptoms such as fever, infection of bat immune cells in bat-mice, interferon production, inflammation activation and other immune markers are measured and compared between humanized mice and bat-mice. E. spelaea infected with Melaka virus is the negative control for this study.

[0099] A bat-mouse model that does not display any clinical symptoms and/or serological evidence for Melaka infection, would be a good model to study other zoonotic virus infection. A screen for suitable infectious agent can be performed, to determine the suitable viruses and the optimum multiplicity of infection (MOI) ratio for each virus. How the bat immune system avoids clinical symptoms can also be investigated.

Example 8

Generation of Bat-Mice and Systemic Lupus Erythematosus (SLE) Model

[0100] To study aspects of autoimmunity, NSG pups within 3 days after birth are sub-lethally irradiated with 1 Gy -ray and transplanted with 1-510.sup.5 bat bone marrow cells via intra-hepatic injection. Bat cell reconstitution is determined at 10-12 weeks post-transplantation by flow cytometry of the peripheral blood. Experimental mice are chosen randomly, regardless of sex and reconstitution level. Pristane [IUPAC name 2,6,10,14-tetramethylpentadecane] (Sigma Aldrich) is injected intra-peritoneally to 12-13 weeks old bat-mice and NSG mice. Levels of bat IgG, IgM, and anti-nuclear (anti-dsDNA, anti-histone, anti-RNP70, anti-SM and anti-SSA) IgGs in the plasma of control and pristane-injected bat-mice are measured using ELISA quantification kits according to manufacturer's instruction (From Bethyl laboratories, and Alpha Diagnostic Inc, respectively).

Histopathology and Immunohistochemistry

[0101] Lungs and kidneys from control and pristane-injected bat-mice and NSG mice are harvested at eight weeks post-injection, fixed with 10% formalin and embedded in paraffin for processing into 5 m tissue sections. Rehydrated lung and kidney sections are stained with Hematoxylin & Eosin (H&E) (Thermo Scientific), and evaluated by a pathologist who is blinded to the samples' identities. Glomerular enlargement is quantified by area measurement of 50 random glomeruli from kidneys of each experimental animal. Images are captured using Brightfield slide scanner (Axio Scan Z1) and processed by Zen software (Carl Zeiss). For immunohistochemistry (IHC), kidney sections are subjected to heat-mediated antigen retrieval with sodium citrate (pH 6) buffer prior to staining with appropriate antibodies. IHC staining is performed using the SuperPicture 3rd Gen IHC Detection Kit (Life Technologies) according to manufacturer's instruction. Primary antibodies used in the study include anti-human IgG, anti-human IgM (Bethyl Laboratories), and anti-human CD45 (AbCAM). Anti-mouse, anti-rabbit and anti-goat HRP-conjugated secondary antibodies are purchased from Life Technologies.

Example 9

Stress Stimulation

[0102] While bats are known to carry multiple zoonotic viruses that are fatal to human, without displaying any clinical symptoms, less is known about bacterial infection in bats. To understand if the bat's immune system has a differential response to bacterial infection as compared to viral infection, both P. alecto and E. spelaea are injected with stimulants such as lipopolysaccharide and CpG oligodeoxynucleotides to mimic bacterial infection. Treated P. alecto and E. spelaea are euthanized 12 hours post-injection. Blood, peritoneal lavage, lung lavage, lung, liver, kidney, spleen and intestinal tissue are harvested from the bats. A portion of the tissues from each organ is frozen for proteomic works and a portion is frozen later for RNA expression studies. Lastly, the remaining tissues are processed for downstream analyses such as FACS and histology staining as described in the methods herein. The proteomic and RNA expression data from stimulated bats are compared to non-stimulated bats to determine the changes in the cytokine and chemokines profiles. The changes in the immune cell subsets upon stimulation can be identified via antibody staining and flow cytometry. In addition, these data will also be compared to data from human cells stimulated with the same stimulants. The differences between stimulated bat and human cells can help understand the immune dampening within the bat immune system. Lastly, the data from this study would form the baseline for future bat-mice studies to determine which cytokines are upregulated or downregulated in response to stress.

[0103] The novel bat-mouse model provided herein represents a major technical advancement and will accelerate many aspects of bat research. The bat-mouse platform provides a genetically consistent model for carrying out in vivo studies on bat immunity, as well as for other biological research.

[0104] Any listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that such document is part of the state of the art or is common general knowledge.

REFERENCES

[0105] Ahn, M., Cui, J., Irving, A. T. & Wang, L.-F. Unique loss of the PYHIN gene family in bats amongst mammals: implications for inflammasome sensing. Sci. Rep. 6; 21722 (2016). [0106] Drake, A. C., Chen, Q. & Chen, J. Engineering humanized mice for improved hematopoietic reconstitution. Cell. Mol. lmmunol. 9; 215-224 (2012). [0107] Green, M. R., Sambrook, J. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press (2012). [0108] Korngold, R. & Sprent, J. Lethal graft-versus-host disease after bone marrow transplantation across minor histocompatibility barriers in mice. Prevention by removing mature T cells from marrow. J. Exp. Med. 148; 1687-1698 (1978). [0109] Olival, K. J., Hosseini, P. R., Zambrana-Torrelio, C., Ross, N., Bogich, T. L., & Daszak, P., Host and viral traits predict zoonotic spillover from mammals. Nature 546; 646-650 (2017). [0110] Ploemacher, R. E., van der Sluijs, J. P., Voerman, J. S. & Brons, N. H. An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 74; 2755-2763 (1989). [0111] Reddy, P., Negrin, R. & Hill, G. R. Mouse models of bone marrow transplantation. Biol. Blood Marrow Transplant. 14; 129-135 (2008). [0112] Taswell, C. Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. J. lmmunol. 126; 1614-1619 (1981). [0113] Yong, K. S. M. et al. Human CD34loCD133lo fetal liver cells support the expansion of human CD34hiCD133hi hematopoietic stem cells. Cell. Mol. lmmunol. 13; 605-614 (2016). [0114] Zhang, G. et al. Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339; 456-460 (2013).