Precision mouse models with expanded tropism for human pathogens

ABSTRACT A major limitation of current humanized mouse models is that they primarily enable the analysis of human-specific pathogens that infect hematopoietic cells. However, most human

pathogens target other cell types, including epithelial, endothelial and mesenchymal cells. Here, we show that implantation of human lung tissue, which contains up to 40 cell types,

including nonhematopoietic cells, into immunodeficient mice (lung-only mice) resulted in the development of a highly vascularized lung implant. We demonstrate that emerging and clinically

relevant human pathogens such as Middle East respiratory syndrome coronavirus, Zika virus, respiratory syncytial virus and cytomegalovirus replicate in vivo in these lung implants. When

incorporated into bone marrow/liver/thymus humanized mice, lung implants are repopulated with autologous human hematopoietic cells. We show robust antigen-specific humoral and T-cell

responses following cytomegalovirus infection that control virus replication. Lung-only mice and bone marrow/liver/thymus-lung humanized mice substantially increase the number of human

pathogens that can be studied in vivo, facilitating the in vivo testing of therapeutics. Access through your institution Buy or subscribe This is a preview of subscription content, access

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS METHODOLOGY OF MURINE LUNG CANCER MIMICS CLINICAL LUNG ADENOCARCINOMA

PROGRESSION AND METASTASIS Article Open access 28 February 2025 A ROBUST MOUSE MODEL OF HPIV-3 INFECTION AND EFFICACY OF GS-441524 AGAINST VIRUS-INDUCED LUNG PATHOLOGY Article Open access 05

September 2024 COVID-19 AND GRAFT-VERSUS-HOST DISEASE: A TALE OF TWO DISEASES (AND WHY AGE MATTERS) Article 09 December 2020 DATA AVAILABILITY The data generated are available from

corresponding authors on reasonable request. REFERENCES * Cockrell, A. S. et al. A mouse model for MERS coronavirus-induced acute respiratory distress syndrome. _Nat. Microbiol._ 2, 16226

(2016). Article  CAS  Google Scholar  * Morrison, T. E. & Diamond, M. S. Animal models of Zika virus infection, pathogenesis, and immunity. _J. Virol._ 91, e00009–17 (2017). CAS  PubMed

  PubMed Central  Google Scholar  * Safronetz, D., Geisbert, T. W. & Feldmann, H. Animal models for highly pathogenic emerging viruses. _Curr. Opin. Virol._ 3, 205–209 (2013). Article 

Google Scholar  * Schmitt, K. et al. Zika viral infection and neutralizing human antibody response in a BLT humanized mouse model. _Virology_ 515, 235–242 (2018). Article  CAS  Google

Scholar  * Crawford, L. B., Streblow, D. N., Hakki, M., Nelson, J. A. & Caposio, P. Humanized mouse models of human cytomegalovirus infection. _Curr. Opin. Virol._ 13, 86–92 (2015).

Article  CAS  Google Scholar  * Taylor, G. Animal models of respiratory syncytial virus infection. _Vaccine_ 35, 469–480 (2017). Article  CAS  Google Scholar  * Gupta, U. D. & Katoch, V.

M. Animal models of tuberculosis. _Tuberculosis (Edinb.)_ 85, 277–293 (2005). Article  CAS  Google Scholar  * Fonseca, K. L., Rodrigues, P. N. S., Olsson, I. A. S. & Saraiva, M.

Experimental study of tuberculosis: from animal models to complex cell systems and organoids. _PLoS Pathog._ 13, e1006421 (2017). Article  Google Scholar  * Pickles, R. J. & DeVincenzo,

J. P. Respiratory syncytial virus (RSV) and its propensity for causing bronchiolitis. _J. Pathol._ 235, 266–276 (2015). Article  Google Scholar  * Perlman, R. L. Mouse models of human

disease: an evolutionary perspective. _Evol. Med. Public Health_ 2016, 170–176 (2016). PubMed  PubMed Central  Google Scholar  * Shultz, L. D., Brehm, M. A., Garcia-Martinez, J. V. &

Greiner, D. L. Humanized mice for immune system investigation: progress, promise and challenges. _Nat. Rev. Immunol._ 12, 786–798 (2012). Article  CAS  Google Scholar  * Garcia, J. V.

Humanized mice for HIV and AIDS research. _Curr. Opin. Virol._ 19, 56–64 (2016). Article  Google Scholar  * Wahl, A. et al. A cluster of virus-encoded microRNAs accelerates acute systemic

Epstein–Barr virus infection but does not significantly enhance virus-induced oncogenesis in vivo. _J. Virol._ 87, 5437–5446 (2013). Article  CAS  Google Scholar  * Melkus, M. W. et al.

Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. _Nat. Med._ 12, 1316–1322 (2006). Article  CAS  Google Scholar  * Islas-Ohlmayer, M. et al. Experimental

infection of NOD/SCID mice reconstituted with human CD34+ cells with Epstein–Barr virus. _J. Virol._ 78, 13891–13900 (2004). Article  CAS  Google Scholar  * Bente, D. A., Melkus, M. W.,

Garcia, J. V. & Rico-Hesse, R. Dengue fever in humanized NOD/SCID mice. _J. Virol._ 79, 13797–13799 (2005). Article  CAS  Google Scholar  * Smith, M. S. et al. Granulocyte-colony

stimulating factor reactivates human cytomegalovirus in a latently infected humanized mouse model. _Cell Host Microbe_ 8, 284–291 (2010). Article  CAS  Google Scholar  * Crawford, L. B. et

al. Human cytomegalovirus induces cellular and humoral virus-specific immune responses in humanized BLT mice. _Sci. Rep._ 7, 937 (2017). Article  Google Scholar  * Wang, L. X. et al.

Humanized-BLT mouse model of Kaposi’s sarcoma-associated herpesvirus infection. _Proc. Natl Acad. Sci. USA_ 111, 3146–3151 (2014). Article  CAS  Google Scholar  * Cockrell, A. S. et al.

Mouse dipeptidyl peptidase 4 is not a functional receptor for Middle East respiratory syndrome coronavirus infection. _J. Virol._ 88, 5195–5199 (2014). Article  Google Scholar  * Coleman, C.

M., Matthews, K. L., Goicochea, L. & Frieman, M. B. Wild-type and innate immune-deficient mice are not susceptible to the Middle East respiratory syndrome coronavirus. _J. Gen. Virol._

95, 408–412 (2014). Article  CAS  Google Scholar  * Martinez-Torres, F., Nochi, T., Wahl, A., Garcia, J. V. & Denton, P. W. Hypogammaglobulinemia in BLT humanized mice—an animal model of

primary antibody deficiency. _PloS ONE_ 9, e108663 (2014). Article  Google Scholar  * Nochi, T., Denton, P. W., Wahl, A. & Garcia, J. V. Cryptopatches are essential for the development

of human GALT. _Cell Rep._ 3, 1874–1884 (2013). Article  CAS  Google Scholar  * Dudek, T. E. et al. Rapid evolution of HIV-1 to functional CD8(+) T cell responses in humanized BLT mice.

_Sci. Transl. Med._ 4, 143ra198 (2012). Article  Google Scholar  * Brainard, D. M. et al. Induction of robust cellular and humoral virus-specific adaptive immune responses in human

immunodeficiency virus-infected humanized BLT mice. _J. Virol._ 83, 7305–7321 (2009). Article  CAS  Google Scholar  * Zhou, J., Chu, H., Chan, J. F. & Yuen, K. Y. Middle East respiratory

syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis. _Virol. J._ 12, 218 (2015). Article  Google Scholar  * Frumence, E. et al. The South Pacific

epidemic strain of Zika virus replicates efficiently in human epithelial A549 cells leading to IFN-_β_ production and apoptosis induction. _Virology_ 493, 217–226 (2016). Article  CAS 

Google Scholar  * Liu, S., DeLalio, L. J., Isakson, B. E. & Wang, T. T. AXL-mediated productive infection of human endothelial cells by Zika virus. _Circ. Res._ 119, 1183–1189 (2016).

Article  CAS  Google Scholar  * Hamel, R. et al. Biology of Zika virus infection in human skin cells. _J. Virol._ 89, 8880–8896 (2015). Article  CAS  Google Scholar  * Franks, T. J. et al.

Resident cellular components of the human lung: current knowledge and goals for research on cell phenotyping and function. _Proc. Am. Thorac. Soc._ 5, 763–766 (2008). Article  Google Scholar

  * Sousa, A. Q. et al. Postmortem findings for 7 neonates with congenital Zika virus infection. _Emerg. Infect. Dis._ 23, 1164–1167 (2017). Article  Google Scholar  * Cunha, B. A.

Cytomegalovirus pneumonia: community-acquired pneumonia in immunocompetent hosts. _Infect. Dis. Clin. North Am._ 24, 147–158 (2010). Article  Google Scholar  * Gordon, C. L. et al. Tissue

reservoirs of antiviral T cell immunity in persistent human CMV infection. _J. Exp. Med._ 214, 651–667 (2017). CAS  PubMed  PubMed Central  Google Scholar  * Lyon, S. M. & Rossman, M. D.

Pulmonary tuberculosis. _Microbiol. Spectr._ 5, 1–13 (2017). Article  Google Scholar  * Gessner, R. et al. High-resolution, high-contrast ultrasound imaging using a prototype dual-frequency

transducer: in vitro and in vivo studies. _IEEE Trans. Ultrason. Ferroelectr. Freq. Control_ 57, 1772–1781 (2010). Article  Google Scholar  * Gessner, R. C., Frederick, C. B., Foster, F. S.

& Dayton, P. A. Acoustic angiography: a new imaging modality for assessing microvasculature architecture. _International J. Biomed. Imaging_ 2013, 936593 (2013). Article  Google Scholar

  * Davis, L. S. et al. Inflammation, immune reactivity, and angiogenesis in a severe combined immunodeficiency model of rheumatoid arthritis. _Am. J. Pathol._ 160, 357–367 (2002). Article 

Google Scholar  * Huang, S. Y., Tien, H. F., Su, F. H. & Hsu, S. M. Nonirradiated NOD/SCID-human chimeric animal model for primary human multiple myeloma: a potential in vivo culture

system. _Am. J. Pathol._ 164, 747–756 (2004). Article  Google Scholar  * Hou, W. et al. Determination of the cell permissiveness spectrum, mode of RNA replication, and RNA-protein

interaction of Zika virus. _BMC Infect. Dis._ 17, 239 (2017). Article  Google Scholar  * Liesman, R. M. et al. RSV-encoded NS2 promotes epithelial cell shedding and distal airway

obstruction. _J. Clin. Invest._ 124, 2219–2233 (2014). Article  CAS  Google Scholar  * Aherne, W., Bird, T., Court, S. D., Gardner, P. S. & McQuillin, J. Pathological changes in virus

infections of the lower respiratory tract in children. _J. Clin. Pathol._ 23, 7–18 (1970). Article  CAS  Google Scholar  * Arend, K. C., Ziehr, B., Vincent, H. A. & Moorman, N. J.

Multiple transcripts encode full-length human cytomegalovirus IE1 and IE2 proteins during lytic infection. _J. Virol._ 90, 8855–8865 (2016). Article  CAS  Google Scholar  * Azevedo, L. S. et

al. Cytomegalovirus infection in transplant recipients. _Clinics (Sao Paulo)_ 70, 515–523 (2015). Article  Google Scholar  * Sathaliyawala, T. et al. Distribution and compartmentalization

of human circulating and tissue-resident memory T cell subsets. _Immunity_ 38, 187–197 (2013). Article  CAS  Google Scholar  * Pitt, E. A. et al. The D-form of a novel heparan binding

peptide decreases cytomegalovirus infection in vivo and in vitro. _Antiviral Res._ 135, 15–23 (2016). Article  CAS  Google Scholar  * Sinzger, C. et al. Cloning and sequencing of a highly

productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E. _J. Gen. Virol._ 89, 359–368 (2008). Article  CAS  Google Scholar  * Ostermann, E., Spohn, M.,

Indenbirken, D. & Brune, W. Complete genome sequence of a human cytomegalovirus strain AD169 bacterial artificial chromosome clone. _Genome Announc._ 4, 1–2 (2016). Article  Google

Scholar  * Wang, D. & Shenk, T. Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism. _J. Virol._ 79, 10330–10338 (2005). Article  CAS  Google Scholar 

* Munro, S. C. et al. Diagnosis of and screening for cytomegalovirus infection in pregnant women. _J. Clin. Microbiol._ 43, 4713–4718 (2005). Article  CAS  Google Scholar  * Gamadia, L. E.,

Rentenaar, R. J., van Lier, R. A. & ten Berge, I. J. Properties of CD4(+) T cells in human cytomegalovirus infection. _Hum. Immunol._ 65, 486–492 (2004). Article  CAS  Google Scholar  *

Vaz-Santiago, J. et al. Ex vivo stimulation and expansion of both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells of human cytomegalovirus-seropositive blood donors by

using a soluble recombinant chimeric protein, IE1-pp65. _J. Virol._ 75, 7840–7847 (2001). Article  CAS  Google Scholar  * Sinclair, E. et al. CMV antigen-specific CD4+ and CD8+ T cell IFNγ

expression and proliferation responses in healthy CMV-seropositive individuals. _Viral Immunol._ 17, 445–454 (2004). Article  CAS  Google Scholar  * Gillespie, G. M. et al. Functional

heterogeneity and high frequencies of cytomegalovirus-specific CD8(+) T lymphocytes in healthy seropositive donors. _J. Virol._ 74, 8140–8150 (2000). Article  CAS  Google Scholar  * Nastke,

M. D. et al. Major contribution of codominant CD8 and CD4 T cell epitopes to the human cytomegalovirus-specific T cell repertoire. _Cell. Mol. Life Sci._ 62, 77–86 (2005). Article  CAS 

Google Scholar  * Kessing, C. F. et al. In vivo suppression of HIV rebound by didehydro-cortistatin a, a “block-and-lock” strategy for HIV-1 treatment. _Cell Rep._ 21, 600–611 (2017).

Article  CAS  Google Scholar  * Kovarova, M. et al. A long-acting formulation of the integrase inhibitor raltegravir protects humanized BLT mice from repeated high-dose vaginal HIV

challenges. _J. Antimicrob. Chemother._ 71, 1586–1596 (2016). Article  CAS  Google Scholar  * Tsai, P. et al. CD19xCD3 DART protein mediates human B-cell depletion in vivo in humanized BLT

mice. _Mol. Ther. Oncolytics_ 3, 15024 (2016). Article  CAS  Google Scholar  * Lilleri, D., Zelini, P., Fornara, C., Comolli, G. & Gerna, G. Inconsistent responses of

cytomegalovirus-specific T cells to pp65 and IE-1 versus infected dendritic cells in organ transplant recipients. _Am. J. Transplant._ 7, 1997–2005 (2007). Article  CAS  Google Scholar  *

Jackson, S. E., Mason, G. M., Okecha, G., Sissons, J. G. & Wills, M. R. Diverse specificities, phenotypes, and antiviral activities of cytomegalovirus-specific CD8+ T cells. _J. Virol._

88, 10894–10908 (2014). Article  CAS  Google Scholar  * Braendstrup, P. et al. Identification and HLA-tetramer-validation of human CD4+ and CD8+ T cell responses against HCMV proteins IE1

and IE2. _PloS ONE_ 9, e94892 (2014). Article  Google Scholar  * Khan, N., Cobbold, M., Keenan, R. & Moss, P. A. Comparative analysis of CD8+ T cell responses against human

cytomegalovirus proteins pp65 and immediate early 1 shows similarities in precursor frequency, oligoclonality, and phenotype. _J. Infect. Dis._ 185, 1025–1034 (2002). Article  CAS  Google

Scholar  * Sacre, K. et al. Expansion of human cytomegalovirus (HCMV) immediate-early 1-specific CD8+ T cells and control of HCMV replication after allogeneic stem cell transplantation. _J.

Virol._ 82, 10143–10152 (2008). Article  CAS  Google Scholar  * Honeycutt, J. B. et al. Macrophages sustain HIV replication in vivo independently of T cells. _J. Clin. Invest._ 126,

1353–1366 (2016). Article  Google Scholar  * Olesen, R. et al. ART influences HIV persistence in the female reproductive tract and cervicovaginal secretions. _J. Clin. Invest._ 126, 892–904

(2016). Article  Google Scholar  * Shelton, S. E. et al. Quantification of microvascular tortuosity during tumor evolution using acoustic angiography. _Ultrasound Med. Biol._ 41, 1896–1904

(2015). Article  Google Scholar  * Honeycutt, J. B. et al. HIV persistence in tissue macrophages of humanized myeloid-only mice during antiretroviral therapy. _Nat. Med._ 23, 638–643 (2017).

Article  CAS  Google Scholar  * Fulcher, M. L., Gabriel, S., Burns, K. A., Yankaskas, J. R. & Randell, S. H. Well-differentiated human airway epithelial cell cultures. _Methods Mol.

Med._ 107, 183–206 (2005). CAS  PubMed  Google Scholar  * Zhang, S. et al. Neutralization mechanism of a highly potent antibody against Zika virus. _Nat. Commun._ 7, 13679 (2016). Article 

CAS  Google Scholar  * Lanciotti, R. S. et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. _Emerg. Infect. Dis._ 14, 1232–1239

(2008). Article  CAS  Google Scholar  * Hallak, L. K., Spillmann, D., Collins, P. L. & Peeples, M. E. Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection.

_J. Virol._ 74, 10508–10513 (2000). Article  CAS  Google Scholar  * O’Connor, C. M. & Shenk, T. Human cytomegalovirus pUS27 G protein-coupled receptor homologue is required for efficient

spread by the extracellular route but not for direct cell-to-cell spread. _J. Virol._ 85, 3700–3707 (2011). Article  Google Scholar  * Wang, D., Bresnahan, W. & Shenk, T. Human

cytomegalovirus encodes a highly specific RANTES decoy receptor. _Proc. Natl Acad. Sci. USA_ 101, 16642–16647 (2004). Article  CAS  Google Scholar  * Ziehr, B., Lenarcic, E., Cecil, C. &

Moorman, N. J. The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication. _Virology_ 489, 194–201 (2016). Article  CAS  Google Scholar  * Goonetilleke, N. et

al. Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified

vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes. _J. Virol._ 80, 4717–4728 (2006). Article  CAS  Google Scholar  Download references

ACKNOWLEDGEMENTS This work was supported by NIH grants no. AI103311 (N.J.M.), no. AI123811 (N.J.M.), no. AI110700 (R.S.B.), no. AI100625 (R.S.B.), no. P30 AI027763 (N.G.), no. AI113736

(R.J.P.), no. T32 HL069768 (I.G.N.), no. AI123010 (A.W.), no. AI111899 (J.V.G.), no. AI140799 (J.V.G.), no. MH108179 (J.V.G.), no. CA189479 (P.A.D.) and no. CA170665 (P.A.D) and the North

Carolina University Cancer Research Fund (N.J.M.). This work was also supported by the UNC Center for AIDS Research (CFAR) (grant no. P30 AI050410). We thank K. Arend for the generation of

HCMV virus stocks used in these experiments. We thank P. Collins and M. Peeples for recombinant RSV expressing GFP and C. O’Connor for recombinant HCMV TB40/E expressing luciferase. We thank

G. Clutton for input on the analysis of antigen-specific T-cell responses. The authors thank members of the Garcia laboratory for technical assistance. We thank technicians at the UNC

Animal Histopathology Core, The Marsico Lung Institute Tissue and Procurement Core, and the Department of Comparative Medicine. We also thank J. Schmitz and technicians at the UNC Clinical

Microbiology/Immunology Laboratories. We thank J. Nelson and technicians at the UNC CFAR Virology Core Laboratory and K. Mollan at the UNC CFAR Biostatistics Core. The authors thank M.T.

Heise, L.J. Picker and J.P. Ting for manuscript advice and helpful discussions. AUTHOR INFORMATION Author notes * Christian R. Aguilera-Sandoval Present address: BD Life Sciences, San Jose,

CA, USA * These authors contributed equally: Chandrav De, Maria Abad Fernandez. AUTHORS AND AFFILIATIONS * Division of Infectious Diseases, International Center for the Advancement of

Translational Science, Center for AIDS Research, University of North Carolina, School of Medicine, Chapel Hill, NC, USA Angela Wahl, Chandrav De, Rachel A. Cleary, Claire E. Johnson, 

Nathaniel J. Schramm, Christian R. Aguilera-Sandoval & J. Victor Garcia * Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC, USA Maria Abad

Fernandez, Erik M. Lenarcic, Yinyan Xu, Laura M. Rank, Heather A. Vincent, Wes Sanders, Allison Boone, Ralph S. Baric, Raymond J. Pickles, Miriam Braunstein, Nathaniel J. Moorman & Nilu

Goonetilleke * Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA Erik M. Lenarcic, Heather A. Vincent, Wes Sanders & Nathaniel J. Moorman *

Department of Epidemiology, University of North Carolina, Chapel Hill, NC, USA Adam S. Cockrell & Ralph S. Baric * Joint Department of Biomedical Engineering, University of North

Carolina and North Carolina State University, Chapel Hill, NC, USA Isabel G. Newsome & Paul A. Dayton * Marsico Lung Institute, University of North Carolina, Chapel Hill, NC, USA Allison

Boone & Raymond J. Pickles * Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA William H. Hildebrand * UNC HIV Cure

Center, University of North Carolina, Chapel Hill, NC, USA Nilu Goonetilleke Authors * Angela Wahl View author publications You can also search for this author inPubMed Google Scholar *

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also search for this author inPubMed Google Scholar CONTRIBUTIONS A.W. inoculated mice with HCMV; collected and processed PB and tissues from HCMV-exposed mice; performed experiments with PB

and tissue cells and analyzed the data; performed the flow cytometric and IHC analysis of naïve LoM and BLT-L mice; performed the human cytokine/chemokine analysis; inoculated mice with BCG

and collected tissues from BCG-exposed mice; conceived and designed experiments; contributed to data interpretation, data presentation and manuscript writing; and conceived, designed and

coordinated the study, the preparation of the manuscript and its revision. C.D. inoculated mice with HCMV; performed the GCV experiment, the in vivo imaging of mice and neutralization assay;

collected and processed PB and tissues from HCMV-exposed mice; performed experiments with PB and tissue cells and analyzed the data; performed the human cytokine/chemokine analysis;

inoculated mice with RSV; and collected and processed tissues and measured RSV-GFP expression by flow cytometry. M.A.F. performed experiments with PB and tissue cells and analyzed the data;

standardized, designed, performed and analyzed immunological assays; and contributed to data interpretation, data presentation and manuscript writing. E.M.L. performed experiments with PB

and tissue cells and analyzed the data and performed the HCMV real-time PCR analyses. Y.X. performed experiments with PB and tissue cells and analyzed the data and standardized, designed,

performed and analyzed immunological assays. A.S.C. produced stocks of MERS-CoV; inoculated mice with MERS-CoV and collected tissues from MERS-CoV infected animals; conceived and designed

experiments; and contributed to data interpretation, data presentation and manuscript writing. R.A.C. performed dual immunofluorescence staining of infected LoM. C.E.J. performed

immunofluorescence staining of animals, collected tissues from BCG-exposed mice and performed the acid-fast staining. N.J.S. produced stocks of ZIKV, inoculated mice with ZIKV, and collected

and processed tissue from ZIKV-infected mice. L.M.R. processed and cultured tissue homogenate to determine the number of the BCG c.f.u. and the analyzed data. I.G.N. performed the

ultrasound and acoustic angiography imaging and analysis. H.A.V. and W.S. performed experiments with PB and tissue cells and analyzed the data and performed the HCMV transcriptome analysis.

C.R.A.S. collected and processed PB and tissues from HCMV-exposed mice. A.B. performed immunofluorescence staining of animals. W.H.H. helped conceive and design experiments. P.A.D., R.S.B.,

N.J.M. and N.G. conceived and designed experiments and contributed to data interpretation, data presentation and manuscript writing. R.J.P. provided stocks of RSV-GFP; performed the

immunofluorescent analysis of tissues sections for RSV infection and AB-PAS staining, and the analysis of human lung implant structures; conceived and designed experiments; and contributed

to data interpretation, data presentation and manuscript writing. M.B. processed and cultured tissue homogenate to determine the number of the BCG c.f.u. and the analyzed data; conceived and

designed experiments; and contributed to data interpretation, data presentation and manuscript writing. J.V.G. conceived and designed experiments and contributed to data interpretation,

data presentation and manuscript writing; and conceived, designed and coordinated the study, the preparation of the manuscript and its revision. CORRESPONDING AUTHORS Correspondence to

Angela Wahl or J. Victor Garcia. ETHICS DECLARATIONS COMPETING INTERESTS P.A.D. is an inventor of the acoustic angiography imaging technique, and a cofounder of SonoVol, Inc., a company that

has licensed this patent. ADDITIONAL INFORMATION PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 HISTOLOGICAL ANALYSIS OF THE STRUCTURE AND CELLULAR COMPOSITION OF DONOR MATCHED HUMAN LUNG TISSUE PRE- AND POST-IMPLANTATION. The

structure and cellular composition of human lung tissue pre-implantation (n=2 analyzed) and donor matched LoM lung implant harvested 2 months post-implantation (n=4 analyzed) were analyzed

by (a) H&E and (b) immunofluorescent staining. In b, co-staining was performed for epithelial cells (cytokeratin 19, magenta) and cilia (alpha-acetylated tubulin, green) or club cells

(CC10, green). Arrows show cuboidal cells lining alveoli. In a, scale bars are 100 µm (left panel) and 200 µm (right panel). In b, scale bars are 200 µm (left panels) and 100 µm (right

panels). SUPPLEMENTARY FIGURE 2 PRESENCE OF MOUSE CELLS IN THE HUMAN LUNG IMPLANTS OF LOM AND NSG MOUSE LUNG. Immunohistochemical staining for murine epithelial cells, endothelial cells,

hematopoietic cells in LoM human lung implants (n=3 analyzed, top panels) and the mouse lung (n=1 analyzed, bottom panels). Images: 10X, scale bars: 100 µm, positive cells: brown. m, mouse.

SUPPLEMENTARY FIGURE 3 PRESENCE OF HUMAN IMMUNE CELLS IN HUMAN LUNG TISSUE PRE- AND POST-IMPLANTATION. Immunohistochemical staining for human hematopoietic (hCD45) cells including

macrophages (hCD68), dendritic cells (hCD11c), B cells (hCD20) and T cells (hCD3) in human donor matched lung tissue pre-implantation (n=1 analyzed, left panels) and two months

post-implantation (n=1 analyzed, right panels). Images: 10X, scale bars: 100 µm, positive cells: brown. SUPPLEMENTARY FIGURE 4 IN VIVO GENE EXPRESSION PROFILE OF HCMV-INFECTED LOM IS

CONSISTENT WITH LYTIC REPLICATION. Total RNA was extracted from human lung implants harvested from HCMV TB40/E infected LoM 14 days post exposure (n=2 TB40/E infected implants). Double

stranded cDNA ((ds)cDNA) was generated from ribosomal RNA (rRNA) depleted total RNA. HCMV (ds)cDNA was enriched with custom designed biotinylated probes spanning both strands of the entire

HCMV genome and sequenced using next generation sequencing. High quality reads were aligned to the HCMV genome, and viral expression was quantified in read per kilobase per million (rpkm).

Values show read counts per gene normalized to gene length read (rpkm). SUPPLEMENTARY FIGURE 5 RECONSTITUTION OF THE PERIPHERAL BLOOD OF BLT-L MICE WITH HUMAN INNATE AND ADAPTIVE IMMUNE

CELLS. Levels of (a) human hematopoietic cells (hCD45) including (b) human myeloid cells (hCD33), B cells (hCD19) and T cells (hCD3) as well as the (c) levels of CD4+ (hCD4) and CD8+ (hCD8)

T cells and (d) ratio of human CD4:CD8 T cells in the peripheral blood of BLT-L mice (n=11, filled circles). Horizontal lines represent mean ± s.e.m. SUPPLEMENTARY FIGURE 6 LEVELS OF HUMAN

IMMUNE CELLS IN THE HUMAN LUNG IMPLANTS AND MOUSE LUNG OF BLT-L MICE. Levels of (a) human hematopoietic cells (hCD45) including (b) human myeloid cells (hCD33), B cells (hCD19) and T cells

(hCD3) in the human lung implants (circles; hCD45, hCD33, and hCD3 n=18, hCD19 n=15) and mouse lung (squares, n=11) of BLT-L mice. (c) Levels of CD4+ (hCD4) and CD8+ (hCD8) T cells and (d)

ratio of human CD4:CD8 T cells in the human lung implants (circles, n=15) and mouse lungs (squares, n=11) of BLT-L mice. (e) Human CD4+ and CD8+ T cell activation (CD38+HLA-DR+) levels in

the human lung implant (circles, n=7) and mouse lung (squares, n=4) of BLT-L mice. Horizontal lines represent mean ± s.e.m. Human immune cell levels in the human lung implants and mouse lung

were compared with a two-tailed Mann-Whitney test. SUPPLEMENTARY FIGURE 7 SYSTEMIC PRESENCE OF HUMAN IMMUNE CELLS IN BLT-L MICE. (a-c) The memory phenotype of human T cells in the human

lung implants of BLT-L mice (n=4 BLT-L mice, one lung implant per animal). (a) Percent of CD4+ (filled circles) and CD8+ (filled squares) human T cells expressing a memory phenotype

(CD45RO+). (b) Percent of memory (CD45RO+) CD4+ (circles) and CD8+ (squares) human T cells expressing an effector memory (Tem, CCR7neg, closed symbols) or central memory (Tcm, CCR7+, open

symbols) phenotype. (c) Percent of memory (CD45RO+) CD4+ (filled circles) and CD8+ (filled squares) T cells that are tissue-resident (TRM, CD69+). (d) Flow cytometry gating scheme. Regions

identify the following human cell populations: RI (live cells), RII (human hematopoietic cells), RIII (T cells), RIV (CD8+ T cells), RV (CD4+ T cells), RVI (memory CD8+ T cells), RVII (CD8+

Tem), RVIII (CD8+ Tcm), RIX (CD8+ TRM), RX (memory CD4+ T cells), RXI (CD4+ Tem), RXII (CD4+ Tcm) and RXIII (CD4+ TRM). In a-c, horizontal lines represent mean ± s.e.m. (e) Human

hematopoietic (hCD45) cells including dendritic cells (hCD11c), macrophages (hCD68), B cells (hCD20) and T cells (hCD3, hCD4 and hCD8) in lymphoid (spleen and lymph nodes) and non-lymphoid

(liver and mouse lung) of BLT-L mice by immunohistochemical staining (positive cells: brown). Images shown are at 20X magnification and represent three BLT-L mice (scale bars: 100 µm).

SUPPLEMENTARY FIGURE 8 INCREASED PLASMA HUMAN CYTOKINE AND CHEMOKINE LEVELS IN BLT-L MICE FOLLOWING HCMV EXPOSURE. Levels of human GM-CSF IFN-γ, IL-6, IL-8, MDC, IP-10, GRO and MCP-1 in the

PB plasma of BLT-L mice (n=10 mice, filled circles) pre and 4 days after HCMV TB40/E inoculation. A value of 3.2 pg/ml was graphed for measurements below the limit of detection of the assay

(3.2 pg/ml, shown with a dashed line). Human cytokine and chemokine levels pre and post HCMV inoculation were compared with a two-tailed Wilcoxon matched-pairs signed rank test.

SUPPLEMENTARY FIGURE 9 IN VIVO REPLICATION OF HCMV AD169 IN LOM HUMAN LUNG IMPLANTS. (a) HCMV-DNA levels in LoM human lung implants at 4, 7, 14, 21 and 28 days post AD169 exposure (day 4:

n=3 implants, days 7, 14, 21 and 28: n=4 implants, filled squares). Horizontal lines represent mean ± s.e.m. (b) HCMV immediate early (IE), early (E) and late (L) proteins in the human lung

implant of an AD169-infected LoM 21 days post-exposure (n=1 lung implant analyzed, positive cells: brown). Images shown are at 40X magnification (scale bars: 50 µm). Positive cells in the

bottom panel are indicated with black arrows. SUPPLEMENTARY FIGURE 10 PLASMA OF HCMV-EXPOSED BLT-L MICE CONTAINS HCMV NEUTRALIZING ACTIVITY. Heat-inactivated plasma from naïve (n=1, open

blue circles) and repeatedly HCMV TB40/E exposed BLT-L mice (n=6, filled symbols) was incubated with HCMV TB40/E expressing RFP for 1 h prior to the addition of virus to epithelial cells

(ARPE-19). Epithelial cells were incubated at 37 °C with the virus/plasma mixture for 2 h at which time the virus/plasma mixture was removed and fresh media added. Shown is the number of

HCMV TB40/E-RFP+ epithelial cells 72 h post-infection in quadruplicate wells. Horizontal lines represent mean ± s.e.m. The percent reduction in TB40/E RFP+ cells compared to wells infected

with HCMV pre-treated with naïve control plasma is shown in the table. SUPPLEMENTARY FIGURE 11 GATING STRATEGIES FOR THE IDENTIFICATION OF HCMV-SPECIFIC HUMAN T CELL RESPONSES IN BLT-L MICE

BY INTRACELLULAR CYTOKINE STAINING (ICS) AND PENTAMER STAINING. Representative flow cytometry plots indicating the gating used to detect (a) human CD8+ T cells expressing IFN-γ and CD107a

and (b) human CD4+ T cells expressing IFN-γ and TNFα by ICS. (c) Representative flow cytometry plots indicating the gating strategy used to detect HLA class1a-restricted HCMV-specific human

CD8+ T cells by pentamer staining. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–11 and Supplementary Tables 1–10 REPORTING SUMMARY SUPPLEMENTARY VIDEO 1:

VASCULARIZATION OF HUMAN LUNG IMPLANTS Three-dimensional rendering of vascularization (red) of a human lung implant (blue). RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE

CITE THIS ARTICLE Wahl, A., De, C., Abad Fernandez, M. _et al._ Precision mouse models with expanded tropism for human pathogens. _Nat Biotechnol_ 37, 1163–1173 (2019).

https://doi.org/10.1038/s41587-019-0225-9 Download citation * Received: 04 December 2018 * Accepted: 12 July 2019 * Published: 26 August 2019 * Issue Date: October 2019 * DOI:

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