Animal Resources Center

Galveston, TX, United States

Animal Resources Center

Galveston, TX, United States
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Meyer M.,Galveston National Laboratory | Meyer M.,University of Texas Medical Branch | Garron T.,Galveston National Laboratory | Garron T.,University of Texas Medical Branch | And 15 more authors.
Journal of Clinical Investigation | Year: 2015

Direct delivery of aerosolized vaccines to the respiratory mucosa elicits both systemic and mucosal responses. This vaccine strategy has not been tested for Ebola virus (EBOV) or other hemorrhagic fever viruses. Here, we examined the immunogenicity and protective efficacy of an aerosolized human parainfluenza virus type 3-vectored vaccine that expresses the glycoprotein (GP) of EBOV (HPIV3/EboGP) delivered to the respiratory tract. Rhesus macaques were vaccinated with aerosolized HPIV3/EboGP, liquid HPIV3/EboGP, or an unrelated, intramuscular, Venezuelan equine encephalitis replicon vaccine expressing EBOV GP. Serum and mucosal samples from aerosolized HPIV3/EboGP recipients exhibited high EBOV-specific IgG, IgA, and neutralizing antibody titers, which exceeded or equaled titers observed in liquid recipients. The HPIV3/EboGP vaccine induced an EBOV-specific cellular response that was greatest in the lungs and yielded polyfunctional CD8+ T cells, including a subset that expressed CD103 (αE integrin), and CD4+ T helper cells that were predominately type 1. The magnitude of the CD4+ T cell response was greater in aerosol vaccinees. The HPIV3/EboGP vaccine produced a more robust cell-mediated and humoral immune response than the systemic replicon vaccine. Moreover, 1 aerosol HPIV3/EboGP dose conferred 100% protection to macaques exposed to EBOV. Aerosol vaccination represents a useful and feasible vaccination mode that can be implemented with ease in a filovirus disease outbreak situation. © 2015, American Society for Clinical Investigation. All rights reserved.


Generation of Ptpn11E76K-neo/+mice have previously been reported5. A neo cassette with a stop codon flanked by loxP sites was inserted in the second intron of the Ptpn11 allele followed by the mutation GAA (E) to AAA (K) at the amino acid 76 encoding position in the third exon. The mice were backcrossed to C57BL/6 mice for more than 10 generations. Ptpn11D61G/+ mice4 were originally imported from Beth Israel Deaconess Medical Center. Nestin-Cre+30, Mx1-Cre+31, Vav1-Cre+32, Prx1-Cre+33, Lepr-Cre+34, Osx1-Cre+35, Oc-Cre+36, and VE-Cadherin-Cre+-ERT2 (ref. 37) transgenic mice used in this study were purchased from the Jackson Laboratory or obtained from the investigators who originally developed the mouse lines. Mice of the same age, sex, and genotype were mixed and then randomly grouped for subsequent analyses (investigators were not blinded during allocation, during experiments and outcome assessment). All mice were kept under specific-pathogen-free conditions in the Animal Resources Center at Case Western Reserve University and subsequently Emory University Division of Animal Resources. All animal procedures complied with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Ptpn11E76K/+Mx1-Cre+ mice and Ptpn11+/+Mx1-Cre+ littermates (8 weeks old) were administered with i.p. injection of 3 doses of pI–pC (1.0 μg per g body weight) every other day over 5 days. Ptpn11E76K/+VE-Cadherin-Cre+-ERT2 mice and Ptpn11+/+VE-Cadherin-Cre+-ERT2 littermates (4–6 weeks old) were administered with i.p. injection of 3 doses of tamoxifen (9.0 mg per 40 g body weight) every other day over 5 days. Mice were analysed at the indicated time points after pI–pC or tamoxifen administration. Acute leukaemia progression in pI–pC administered Ptpn11E76K/+Mx1-Cre+ and Ptpn11E76K/+Vav1-Cre+ mice was determined as we previously described5. No statistical methods were used to predetermine sample size. De-identified BM biopsies from PTPN11-mutation-positive Noonan syndrome patients with JMML or non-syndromic PTPN11 mutation-positive patients with JMML were obtained from the University of California, San Francisco Tissue Cancer Cell Bank and Children’s Healthcare of Atlanta, Emory University. Informed consent was obtained from all subjects. The experiments involving human subjects were reviewed and approved (Exemption IV) by the Institutional Review Board of Emory University. BM cells (2 × 106) collected from indicated donor mice were transplanted into lethally irradiated (1,100 cGy) recipient mice with the indicated genotypes through tail vein injection. Recipients were monitored for MPN development for 6–8 months. To determine the abundance of the neo cassette in the targeted Ptpn11 allele, genomic DNA of haematopoietic cells, MSPCs, or other indicated cells was extracted with a ZR-Duet DNA/RNA MiniPrep extraction kit (Zymo Research). The abundance of the neo cassette was then quantified by qPCR using the Applied Biosystems 7500 Fast Real-Time PCR System. The PCR primers used were: 5′-TGGGAAGACAATAGCAGGCA-3′ and 5′-CCCACTCACCTTGTCATGTA-3′. The pool size, cell cycle status, apoptosis, and cell signalling activities of HSCs were analysed by multiparameter FACS analyses, as previously described38. In brief, for the HSC-pool-size analysis, fresh BM cells were stained with the following antibodies (eBiosciences, San Diego, unless otherwise noted): lineage antibodies (B220 (RA3-6B2), CD3 (145-2C11), Gr-1 (RB6-8C5), Mac-1 (M1/70), and Ter-119 (TER-119)), anti-Sca-1 (D7, BD Biosciences), anti-c-Kit (2B8), anti-CD150 (TC15-12F12.2, BD Biosciences), anti-CD48 (HM48-1), and anti-Flk2 (A2F10.1). Lin−Sca-1+c-Kit+CD150+CD48−Flk2− cells were quantified as HSCs. For the cell cycle analysis, freshly collected BM cells were stained for HSCs as above. Cells were then fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences), stained with Ki-67 antibody, and further incubated with Hoechest 33342 (20 μg ml−1). For the apoptosis analysis, BM cells were stained for HSCs, and then incubated with Annexin V and 7-amino-actinomycin D (BD Biosciences). For cell signalling analyses, BM cells were stained for HSCs, fixed and permeabilized using a Cytofix/Cytoperm kit, and then stained with anti-phospho-Erk (mouse IgG) (E-4, Santa Cruz Biotechnology), anti-phospho-Akt (rabbit IgG) (C31E5E, Cell Signaling), or anti-phospho-NF-κB (rabbit IgG) (93H1, Cell Signaling) antibodies, washed and further incubated with AlexaFluor488-conjugated secondary antibodies (goat anti-mouse IgG or goat anti-rabbit IgG) (Life technologies). Phosphorylation levels of these signalling proteins were determined by mean fluorescence intensities (MFI) of gated cells. Data were collected on BD LSR II Flow Cytometer (BD Biosciences) and analysed with FlowJo (Treestar). HSCs (Lin−Sca-1+c-Kit+CD150+CD48−Flk2−) sorted from wild-type C57BL/6 mice were cultured in StemSpan medium supplemented with SCF (50 ng ml−1), Flt3 ligand (50 ng ml−1), TPO (50 ng ml−1), IL-3 (20 ng ml−1), and IL-6 (20 ng ml−1) in the presence of IL-1β (10 ng ml−1), CCL3 (20 ng ml−1), CCL4 (20 ng ml−1), or CCL12 (20 ng ml−1). Six days later, cells were collected and analysed for Mac-1+ myeloid cells, F4/80+ macrophages, and CD115+ monocytes. Mouse MSPCs were enriched following a standard protocol39. In brief, BM was collected from long bones. The bones were then crushed and digested with collagenase type II (2.5 mg ml−1) (Worthington Biochemical Corporation). BM cells and digested bone fragments were combined and cultured in DMEM supplemented with 15% fetal bovine serum (FBS). For human MSPC derivation, only BM cells were used. Suspension haematopoietic cells were removed after 24 h. Medium was replenished every 72 h. Colonies of MSPCs appeared 6–8 days after initial plating. To further purify MSPCs, cells were collected and stained with biotin-conjugated CD45 antibody and anti-biotin microbeads. CD45+ haematopoietic cells were depleted using MACS separation columns (Miltenyi Biotec Inc.). The purity of MSPCs (>95%) was further confirmed according to the (CD45−CD140α+Sca-1+) phenotypes39 by multiparameter FACS analyses. For the CFU-F assay, 2 × 106 unfractionated BM cells were plated and cultured for 10–14 days as described above. Cells were stained with 0.5% crystal violet (Sigma-Aldrich) in 10% methanol for 20 min. Colonies formed by more than 50 fibroblast-like cells were counted under a light microscope. For the CFU-GM assay, freshly collected BM cells (2 × 104 cells ml−1) were seeded in 0.9% methylcellulose IMDM medium containing 30% FBS, glutamine (10−4 M), β-mercaptoethanol (3.3 × 10−5 M), and IL-3 (1 ng ml−1) or GM-CSF (1 ng ml−1). After 7 days of culture at 37 °C in a humidified 5% CO incubator, colonies (primarily CFU-GM) formed by more than 50 haematopoietic cells were counted under an inverted microscope. MSPCs (CD45−Ter-119-CD31-CD140α+Sca-1+)39 were freshly isolated from the BM of Ptpn11E76K/+Nestin-Cre+ and Ptpn11+/+Nestin-Cre+ mice. RNA was extracted using the RNeasy Midi kit (Qiagen). Total RNA samples were enriched for polyadenylated transcripts using the Oligotex mRNA Mini kit (Qiagen), and strand-specific RNA-seq libraries were generated using PrepX RNA library preparation kits (IntegenX), following the manufacturer’s protocol. After cleanup with AMPure XP beads (Beckman Coulter) and amplification with Phusion High-Fidelity polymerase (New England BioLabs), RNA libraries were sequenced on a HiSeq 4000 instrument to a depth of at least 20 million reads. The correlation coefficient between the two groups is 0.954, which verifies that the method is accurate (Extended Data Fig. 6b). Before differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the DEGSeq R package (1.12.0). The P values were adjusted using the Benjamini–Hochberg method. Corrected P value of 0.005 and log (fold change) of 1 were set as the threshold for significantly different expression. Femurs were dissected from Ptpn11E76K/+Mx1-Cre+ mice and Ptpn11+/+Mx1-Cre+ littermates 12 weeks after pI–pC administration. BM plasma was collected by flushing one femur with 1.0 ml of phosphate buffered saline (PBS). MSPCs derived from pI–pC-administered Ptpn11E76K/+Mx1-Cre+ and Ptpn11+/+Mx1-Cre+ mice were cultured (4 × 106 cells in 2.0 ml medium) in serum-free DMEM for 48 h. The culture medium was then collected. BM plasma or MSPC culture medium were analysed with Mouse Cytokine Antibody Array blots (R&D Systems) following the instructions provided by the manufacturer. BM plasma collected from one femur and one tibia in 500 μl PBS. Culture medium was collected from mouse MSPCs (4 × 106 cells per 2.0 ml) at second or third passages cultured in serum-free DMEM for 48 h. These samples were assayed for levels of IL-1β and CCL3 using enzyme-linked immunosorbent assay (ELISA) kits (IL-1β: eBioscience; CCL3: R&D Systems) following the instructions provided by the manufacturers. To determine multiple cytokines/chemokines produced by human MSPCs, MSPCs (2 × 104 cells ml−1) were cultured in serum-free StemSpan medium for 72–96 h. To determine multiple protein factors produced by cells from patients with JMML, JMML cells (2 × 105 cells ml−1) were cultured in StemSpan medium supplemented with human SCF (50 ng ml−1), human Flt3 ligand (50 ng ml−1), and human TPO (50 ng ml−1) for 72 h. The culture medium was then collected and cytokine/chemokine levels were determined by the BD Cytometric Bead Array Flex Sets (BD Biosciences) following the manufacturer’s instructions. Human CXCL12 levels in MSPC culture medium were measured using a Human CXCL12/SDF-1 alpha Quantikine ELISA Kit (R&D systems). Frozen tissue sections prepared from 4% paraformaldehyde-fixed and decalcified bones were thawed at room temperature and then rehydrated with PBS. The slides were stained with the following antibodies (eBiosciences, San Diego, unless otherwise noted) following standard procedures: anti-Osteopontin (Abcam), anti-Nestin (MAB353, Millipore), anti-Gr-1 (RB6-8C5), anti-Mac-1 (M1/70), anti-B220 (RA3-6B2), anti-Ter-119 (TER-119), anti-CD3 (145-2C11, BD Biosciences), anti-CD115 (AFS98), anti-CD150 (TC15-12F12.2, BD Biosciences), anti-CD31 (MEC13.3, Biolegend), anti-CD48 (HM48-1), and anti-CD41 (eBioMWReg30) antibodies. Images were acquired using Olympus Confocal Laser Scanning Biological Microscope FV1000 equipped with four lasers ranging from 405 to 635 nm. Images were processed with ImageJ software. Ptpn11E76K/+Osx1-Cre+ mice (6–7 month old) and Ptpn11E76K/+Mx1-Cre+ mice (4 weeks after pI–pC administration) were treated daily via subcutaneous injection with the CCR1 antagonist BX471 ((2R)-1-((2-((aminocarbonyl)amino)-4-chlorophenoxy)acetyl)-4-((4-fluorophenyl)methyl)-2-methylpiperazine) purchased from Tocris Bioscience (50 mg kg−1 of body weight). These animals also received the CCR5 antagonist Maraviroc (4,4-difluoro-N-((S)-3-(3-(3-isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-8-azabicyclo(3.2.1)octan-8-yl)-1-phenylpropyl)cyclohexanecarboxamide) obtained from Selleck Chemicals (0.3 mg ml−1 in the drinking water). Control Ptpn11E76K/+Osx1-Cre+ mice and Ptpn11E76K/+Mx1-Cre+ mice were given vehicle (70% ethanol and 0.5% DMSO for subcutaneous injections, and 1% DMSO in drinking water). Mice were treated for 23 days and then killed for subsequent analyses. Data are presented as mean ± s.d. of all mice analysed in multiple experiments (that is, biological replicates). Statistical significance was determined using unpaired two-tailed Student’s t test. For HSC imaging analyses, two-tier tests were used to first combine technical replicates and then evaluate biological replicates. To determine statistical significance in the incidences of MPN development and malignant progression, Fisher’s exact tests were performed. *P < 0.05; **P < 0.01; ***P < 0.001; N.S., not significant in Extended Data Figs 2, 5.


Filipovska-Naumovska E.,Murdoch University | Abubakar S.M.,Animal Resources Center | Thompson M.J.,Murdoch University | Hopwood D.,Animal Resources Center | And 2 more authors.
Journal of the American Association for Laboratory Animal Science | Year: 2010

A mouse parvovirus (designated MPV1f) was identified in a commercial laboratory mouse colony in Australia. The infection had not been detected by using an rNS1 parvovirus ELISA antigen even though the virus was genetically similar to other MPV1 variants reported previously. A recombinant biotinylated protein based on a truncated VP1 protein of the MPV1 strain was produced and used as antigen for ELISA and Western immunoblots to detect virus infection and determine the seroprevalence of infection in a colony of approximately 45,000 mice. Antibody-positive mice were detected in 8 of 11 rooms sampled, indicating that infection was widespread in the facility. Antibody was detected in 16.2% of 1161 sera obtained from 20 strains of mice. Seroprevalence varied among mouse strains, suggesting genetic variation in the susceptibility of mice to MPV1 or in their antibody response to infection, as has been reported previously in experimentally infected mice. Seroprevalence was high in some inbred strains, including DBA/2JArc and the random-bred strains Hsd:NIH and Arc:Arc(s). Antibody was not detected inC57BL/6J strains, and BALB/c strains showed low seroprevalence of MPV1f. Copyright 2010 by the American Association for Laboratory Animal Science.


Filipovska-Naumovska E.,Murdoch University | Thompson M.J.,Murdoch University | Hopwood D.,Animal Resources Center | Pass D.A.,Animal Resources Center | Wilcox G.E.,Murdoch University
Journal of the American Association for Laboratory Animal Science | Year: 2010

The effect of mouse strain and age at infection on viral replication and concurrent antibody response to mouse parvovirus 1 (isolate MPV1f) was evaluated for 305 d after inoculation in 4 strains of mice. The results confirmed previous reports that mouse strain and age at infection are significant factors in viral persistence and antibody development and detection. Randombred Arc:Arc(s) mice originally bred from CD1 stock inoculated as juveniles (4 wk) or adults (8 wk) developed persistent viral infection for 152 d after inoculation and an antibody response that persisted for 295 d. Mice of C57BL/6J background inoculated as juveniles had detectable viral DNA in large intestinal content and tissues for 24 d after inoculation and an antibody response that persisted for 288 d. However, viral DNA was not detected in tissues of C57BL/6J mice inoculated as adults, although an antibody was detected for 111 d after inoculation; these results suggest probable viral replication in adult C57BL/6J mice but at levels below the limits of detection. BALB/cArc mice inoculated as juveniles or adults had detectable virus DNA in tissues for 108 to 242 d after inoculation, but no antibody was detected. Similarly, BALB/c-Foxn1 nu/ArC mice had detectable levels of viral DNA in tissues for 98 to 131 d but no measurable antibody. The difficulty of detecting antibody in mice with a BALB/c background indicates they are unsuitable for routine surveillance of MPV1f infection. Copyright 2010 by the American Association for Laboratory Animal Science.

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