Advertisement

Current Use of Adenovirus Vectors and Their Production Methods

  • Ekramy E. Sayedahmed
  • Rashmi Kumari
  • Suresh K. MittalEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1937)

Abstract

Various adenovirus (AdV) vector systems have proven to be lucrative options for gene delivery. They can serve as potential vaccine candidates for prevention of several common infectious diseases and hold the promise for gene therapy, especially for cancer. Several AdV vector-based therapies are currently at various stages of clinical trials worldwide, which make an immense interest of both the clinicians and researchers. Since these vectors are easy to manipulate, have broad tropism, and have the capability to yield high titers, this delivery system has a wide range of applications for different clinical settings. This chapter emphasizes on some of the current usages of AdV vectors and their production methods.

Key words

Adenovirus vector Gene delivery system Gene therapy Vector design Vector production Recombinant vaccines 

Notes

Acknowledgments

This work was supported by the Public Health Service grant—AI059374 from the National Institute of Allergy and Infectious Diseases, and the Hatch and Animal Health funds.

References

  1. 1.
    Rowe WP, Huebner RJ, Gilmore LK et al (1953) Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proc Soc Exp Biol Med 84(3):570–573CrossRefGoogle Scholar
  2. 2.
    Enders JF, Bell JA, Dingle JH et al (1956) Adenoviruses: group name proposed for new respiratory-tract viruses. Science 124(3212):119–120CrossRefGoogle Scholar
  3. 3.
    Davison AJ, Benko M, Harrach B (2003) Genetic content and evolution of adenoviruses. J Gen Virol 84(Pt 11):2895–2908.  https://doi.org/10.1099/vir.0.19497-0CrossRefPubMedGoogle Scholar
  4. 4.
    Ewer KJ, Lambe T, Rollier CS et al (2016) Viral vectors as vaccine platforms: from immunogenicity to impact. Curr Opin Immunol 41:47–54.  https://doi.org/10.1016/j.coi.2016.05.014CrossRefPubMedGoogle Scholar
  5. 5.
    Su C (2011) Adenovirus-based gene therapy for cancer. In: Xu K (ed) Viral gene therapy. InTech, Rijeka, p 06.  https://doi.org/10.5772/19757CrossRefGoogle Scholar
  6. 6.
    Pesonen S, Kangasniemi L, Hemminki A (2011) Oncolytic adenoviruses for the treatment of human cancer: focus on translational and clinical data. Mol Pharm 8(1):12–28.  https://doi.org/10.1021/mp100219nCrossRefPubMedGoogle Scholar
  7. 7.
    Tatsis N, Ertl HC (2004) Adenoviruses as vaccine vectors. Mol Ther 10(4):616–629.  https://doi.org/10.1016/j.ymthe.2004.07.013CrossRefPubMedGoogle Scholar
  8. 8.
    Vemula SV, Amen O, Katz JM et al (2013) Beta-defensin 2 enhances immunogenicity and protection of an adenovirus-based H5N1 influenza vaccine at an early time. Virus Res 178(2):398–403.  https://doi.org/10.1016/j.virusres.2013.09.013CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Sharma A, Tandon M, Bangari DS et al (2009) Adenoviral vector-based strategies for cancer therapy. Curr Drug ther 4(2):117–138CrossRefGoogle Scholar
  10. 10.
    Raper SE, Chirmule N, Lee FS et al (2003) Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 80(1–2):148–158CrossRefGoogle Scholar
  11. 11.
    Harvey BG, Worgall S, Ely S et al (1999) Cellular immune responses of healthy individuals to intradermal administration of an E1-E3- adenovirus gene transfer vector. Hum Gene Ther 10(17):2823–2837.  https://doi.org/10.1089/10430349950016555CrossRefPubMedGoogle Scholar
  12. 12.
    Zaiss AK, Machado HB, Herschman HR (2009) The influence of innate and pre-existing immunity on adenovirus therapy. J Cell Biochem 108(4):778–790.  https://doi.org/10.1002/jcb.22328CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Pandey A, Singh N, Vemula SV et al (2012) Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based H5N1 influenza vaccine. PLoS One 7(3):e33428.  https://doi.org/10.1371/journal.pone.0033428CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Mast TC, Kierstead L, Gupta SB et al (2010) International epidemiology of human pre-existing adenovirus (Ad) type-5, type-6, type-26 and type-36 neutralizing antibodies: correlates of high Ad5 titers and implications for potential HIV vaccine trials. Vaccine 28(4):950–957.  https://doi.org/10.1016/j.vaccine.2009.10.145CrossRefPubMedGoogle Scholar
  15. 15.
    Pratt WD, Wang D, Nichols DK et al (2010) Protection of nonhuman primates against two species of Ebola virus infection with a single complex adenovirus vector. Clin Vaccine Immunol 17(4):572–581.  https://doi.org/10.1128/cvi.00467-09CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Wold WS, Toth K (2013) Adenovirus vectors for gene therapy, vaccination and cancer gene therapy. Curr Gene Ther 13(6):421–433CrossRefGoogle Scholar
  17. 17.
    Seregin SS, Amalfitano A (2010) Improving adenovirus based gene transfer: strategies to accomplish immune evasion. Viruses 2(9):2013–2036.  https://doi.org/10.3390/v2092013CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kay MA, Meuse L, Gown AM et al (1997) Transient immunomodulation with anti-CD40 ligand antibody and CTLA4Ig enhances persistence and secondary adenovirus-mediated gene transfer into mouse liver. Proc Natl Acad Sci U S A 94(9):4686–4691CrossRefGoogle Scholar
  19. 19.
    Engelhardt JF, Ye X, Doranz B et al (1994) Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc Natl Acad Sci U S A 91(13):6196–6200CrossRefGoogle Scholar
  20. 20.
    Bangari DS, Mittal SK (2006) Development of nonhuman adenoviruses as vaccine vectors. Vaccine 24(7):849–862.  https://doi.org/10.1016/j.vaccine.2005.08.101CrossRefPubMedGoogle Scholar
  21. 21.
    Mittal SK, Ahi YS, Vemula SV (2016) 19 - xenogenic adenoviral vectors A2 - curiel. In: David T (ed) Adenoviral vectors for gene therapy, 2nd edn. Academic Press, San Diego, pp 495–528.  https://doi.org/10.1016/B978-0-12-800276-6.00019-XCrossRefGoogle Scholar
  22. 22.
    Singh N, Pandey A, Jayashankar L et al (2008) Bovine adenoviral vector-based H5N1 influenza vaccine overcomes exceptionally high levels of pre-existing immunity against human adenovirus. Mol Ther 16(5):965–971.  https://doi.org/10.1038/mt.2008.12CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Moffatt S, Hays J, HogenEsch H et al (2000) Circumvention of vector-specific neutralizing antibody response by alternating use of human and non-human adenoviruses: implications in gene therapy. Virology 272(1):159–167.  https://doi.org/10.1006/viro.2000.0350CrossRefPubMedGoogle Scholar
  24. 24.
    Li X, Bangari DS, Sharma A et al (2009) Bovine adenovirus serotype 3 utilizes sialic acid as a cellular receptor for virus entry. Virology 392(2):162–168.  https://doi.org/10.1016/j.virol.2009.06.029CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Bangari DS, Sharma A, Mittal SK (2005) Bovine adenovirus type 3 internalization is independent of primary receptors of human adenovirus type 5 and porcine adenovirus type 3. Biochem Biophys Res Commun 331(4):1478–1484.  https://doi.org/10.1016/j.bbrc.2005.04.058CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Ehrhardt A, Xu H, Kay MA (2003) Episomal Persistence of Recombinant Adenoviral Vector Genomes during the Cell Cycle In Vivo. J Virol 77(13):7689–7695.  https://doi.org/10.1128/jvi.77.13.7689-7695.2003CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Seiler MP, Cerullo V, Lee B (2007) Immune response to helper dependent adenoviral mediated liver gene therapy: challenges and prospects. Curr Gene Ther 7(5):297–305CrossRefGoogle Scholar
  28. 28.
    Gene Therapy Clinical Trials Worldwide (2017). Available from; http://www.abedia.com/wiley/vectors.php). http://www.abedia.com/wiley/vectors.php
  29. 29.
    Smaill F, Jeyanathan M, Smieja M et al (2013) A human type 5 adenovirus-based tuberculosis vaccine induces robust T cell responses in humans despite preexisting anti-adenovirus immunity. Sci Transl Med 5(205):205ra134.  https://doi.org/10.1126/scitranslmed.3006843CrossRefPubMedGoogle Scholar
  30. 30.
    Vemula SV, Mittal SK (2010) Production of adenovirus vectors and their use as a delivery system for influenza vaccines. Expert Opin Biol Ther 10(10):1469–1487.  https://doi.org/10.1517/14712598.2010.519332CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Ahi YS, Bangari DS, Mittal SK (2011) Adenoviral vector immunity: its implications and circumvention strategies. Curr Gene Ther 11(4):307–320CrossRefGoogle Scholar
  32. 32.
    Zhu J, Huang X, Yang Y (2007) Innate immune response to adenoviral vectors is mediated by both Toll-like receptor-dependent and -independent pathways. J Virol 81(7):3170–3180.  https://doi.org/10.1128/jvi.02192-06CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Sharma A, Tandon M, Ahi YS et al (2010) Evaluation of cross-reactive cell-mediated immune responses among human, bovine and porcine adenoviruses. Gene Ther 17(5):634–642.  https://doi.org/10.1038/gt.2010.1CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Hassan AO, Amen O, Sayedahmed EE et al (2017) Adenovirus vector-based multi-epitope vaccine provides partial protection against H5, H7, and H9 avian influenza viruses. PLoS One 12(10):e0186244.  https://doi.org/10.1371/journal.pone.0186244CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Kim EH, Han GY, Nguyen H (2017) An adenovirus-vectored influenza vaccine induces durable cross-protective hemagglutinin stalk antibody responses in mice. Viruses 9(8):E234.  https://doi.org/10.3390/v9080234CrossRefPubMedGoogle Scholar
  36. 36.
    Kamlangdee A, Kingstad-Bakke B, Anderson TK et al (2014) Broad protection against avian influenza virus by using a modified vaccinia Ankara virus expressing a mosaic hemagglutinin gene. J Virol 88(22):13300–13309.  https://doi.org/10.1128/jvi.01532-14CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Hoelscher MA, Singh N, Garg S et al (2008) A broadly protective vaccine against globally dispersed clade 1 and clade 2 H5N1 influenza viruses. J Infect Dis 197(8):1185–1188.  https://doi.org/10.1086/529522CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Cao W, Liepkalns JS, Hassan AO et al (2016) A highly immunogenic vaccine against A/H7N9 influenza virus. Vaccine 34(6):744–749.  https://doi.org/10.1016/j.vaccine.2015.12.062CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    de Vries RD, Rimmelzwaan GF (2016) Viral vector-based influenza vaccines. Hum Vaccin Immunother 12(11):2881–2901.  https://doi.org/10.1080/21645515.2016.1210729CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Hoelscher MA, Garg S, Bangari DS et al (2006) Development of adenoviral-vector-based pandemic influenza vaccine against antigenically distinct human H5N1 strains in mice. Lancet 367(9509):475–481.  https://doi.org/10.1016/s0140-6736(06)68076-8CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Van Kampen KR, Shi Z, Gao P et al (2005) Safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans. Vaccine 23(8):1029–1036.  https://doi.org/10.1016/j.vaccine.2004.07.043CrossRefPubMedGoogle Scholar
  42. 42.
    Fuchs JD, Bart PA, Frahm N et al (2015) Safety and immunogenicity of a recombinant adenovirus serotype 35-vectored HIV-1 vaccine in adenovirus serotype 5 seronegative and seropositive individuals. J AIDS Clin Res 6(5):461.  https://doi.org/10.4172/2155-6113.1000461CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Milligan ID, Gibani MM, Sewell R et al (2016) Safety and immunogenicity of novel adenovirus type 26- and modified vaccinia ankara-vectored ebola vaccines: a randomized clinical trial. JAMA 315(15):1610–1623.  https://doi.org/10.1001/jama.2016.4218CrossRefPubMedGoogle Scholar
  44. 44.
    O'Hara GA, Duncan CJ, Ewer KJ et al (2012) Clinical assessment of a recombinant simian adenovirus ChAd63: a potent new vaccine vector. J Infect Dis 205(5):772–781.  https://doi.org/10.1093/infdis/jir850CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Osman M, Mistry A, Keding A et al (2017) A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis: first-in-human trial of ChAd63-KH. PLoS Negl Trop Dis 11(5):e0005527.  https://doi.org/10.1371/journal.pntd.0005527CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Ledgerwood JE, DeZure AD, Stanley DA et al (2017) Chimpanzee adenovirus vector ebola vaccine. N Engl J Med 376(10):928–938.  https://doi.org/10.1056/NEJMoa1410863CrossRefPubMedGoogle Scholar
  47. 47.
    Tapia MD, Sow SO, Lyke KE et al (2016) Use of ChAd3-EBO-Z Ebola virus vaccine in Malian and US adults, and boosting of Malian adults with MVA-BN-Filo: a phase 1, single-blind, randomised trial, a phase 1b, open-label and double-blind, dose-escalation trial, and a nested, randomised, double-blind, placebo-controlled trial. Lancet Infect Dis 16(1):31–42.  https://doi.org/10.1016/s1473-3099(15)00362-xCrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Crosby CM, Matchett WE, Anguiano-Zarate SS et al (2017) Replicating single-cycle adenovirus vectors generate amplified influenza vaccine responses. J Virol 91(2):e00720.  https://doi.org/10.1128/jvi.00720-16CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Li Y, Pong RC, Bergelson JM et al (1999) Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy. Cancer Res 59(2):325–330PubMedGoogle Scholar
  50. 50.
    Shen YH, Yang F, Wang H et al (2016) Arg-Gly-Asp (RGD)-modified E1A/E1B double mutant adenovirus enhances antitumor activity in prostate cancer cells in vitro and in mice. PLoS One 11(1):e0147173.  https://doi.org/10.1371/journal.pone.0147173CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Wang H, Li ZY, Liu Y et al (2011) Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14. Nat Med 17(1):96–104.  https://doi.org/10.1038/nm.2270CrossRefPubMedGoogle Scholar
  52. 52.
    Biedermann K, Vogelsang H, Becker I et al (2005) Desmoglein 2 is expressed abnormally rather than mutated in familial and sporadic gastric cancer. J Pathol 207(2):199–206.  https://doi.org/10.1002/path.1821CrossRefPubMedGoogle Scholar
  53. 53.
    Harada H, Iwatsuki K, Ohtsuka M et al (1996) Abnormal desmoglein expression by squamous cell carcinoma cells. Acta Derm Venereol 76(6):417–420PubMedGoogle Scholar
  54. 54.
    Schmitt CJ, Franke WW, Goerdt S et al (2007) Homo- and heterotypic cell contacts in malignant melanoma cells and desmoglein 2 as a novel solitary surface glycoprotein. J Invest Dermatol 127(9):2191–2206.  https://doi.org/10.1038/sj.jid.5700849CrossRefPubMedGoogle Scholar
  55. 55.
    Lee SY, Park HR, Rhee J et al (2013) Therapeutic effect of oncolytic adenovirus expressing relaxin in radioresistant oral squamous cell carcinoma. Oncol Res 20(9):419–425.  https://doi.org/10.3727/096504013x13657689383139CrossRefPubMedGoogle Scholar
  56. 56.
    Vera B, Martinez-Velez N, Xipell E et al (2016) Characterization of the Antiglioma Effect of the Oncolytic Adenovirus VCN-01. PLoS One 11(1):e0147211.  https://doi.org/10.1371/journal.pone.0147211CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Ranki T, Pesonen S, Hemminki A et al (2016) Phase I study with ONCOS-102 for the treatment of solid tumors - an evaluation of clinical response and exploratory analyses of immune markers. J Immunother Cancer 4:17.  https://doi.org/10.1186/s40425-016-0121-5CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Danthinne X, Imperiale MJ (2000) Production of first generation adenovirus vectors: a review. Gene Ther 7(20):1707–1714.  https://doi.org/10.1038/sj.gt.3301301CrossRefPubMedGoogle Scholar
  59. 59.
    Graham FL, Smiley J, Russell WC et al (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36(1):59–74.  https://doi.org/10.1099/0022-1317-36-1-59CrossRefPubMedGoogle Scholar
  60. 60.
    Kamen A, Henry O (2004) Development and optimization of an adenovirus production process. J Gene Med 6(Suppl 1):S184–S192.  https://doi.org/10.1002/jgm.503CrossRefPubMedGoogle Scholar
  61. 61.
    Fallaux FJ, Bout A, van der Velde I et al (1998) New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses. Hum Gene Ther 9(13):1909–1917.  https://doi.org/10.1089/hum.1998.9.13-1909CrossRefPubMedGoogle Scholar
  62. 62.
    Howe JA, Pelka P, Antelman D et al (2006) Matching complementing functions of transformed cells with stable expression of selected viral genes for production of E1-deleted adenovirus vectors. Virology 345(1):220–230.  https://doi.org/10.1016/j.virol.2005.09.029CrossRefPubMedGoogle Scholar
  63. 63.
    Wen S, Schneider DB, Driscoll RM et al (2000) Second-generation adenoviral vectors do not prevent rapid loss of transgene expression and vector DNA from the arterial wall. Arterioscler Thromb Vasc Biol 20(6):1452–1458CrossRefGoogle Scholar
  64. 64.
    Mitani K, Graham FL, Caskey CT et al (1995) Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector. Proc Natl Acad Sci U S A 92(9):3854–3858CrossRefGoogle Scholar
  65. 65.
    Parks R, Evelegh C, Graham F (1999) Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration. Gene Ther 6(9):1565–1573.  https://doi.org/10.1038/sj.gt.3300995CrossRefPubMedGoogle Scholar
  66. 66.
    Alba R, Bosch A, Chillon M (2005) Gutless adenovirus: last-generation adenovirus for gene therapy. Gene Ther 12(Suppl 1):S18–S27.  https://doi.org/10.1038/sj.gt.3302612CrossRefPubMedGoogle Scholar
  67. 67.
    Parks R, Chen L, Anton M et al (1996) A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci U S A 93(24):13565–13570CrossRefGoogle Scholar
  68. 68.
    Hardy S, Kitamura M, Harris-Stansil T et al (1997) Construction of adenovirus vectors through Cre-lox recombination. J Virol 71(3):1842–1849PubMedPubMedCentralGoogle Scholar
  69. 69.
    Vetrini F, Ng P (2010) Gene therapy with helper-dependent adenoviral vectors: current advances and future perspectives. Viruses 2(9):1886–1917.  https://doi.org/10.3390/v2091886CrossRefPubMedPubMedCentralGoogle Scholar
  70. 70.
    Stow ND (1981) Cloning of a DNA fragment from the left-hand terminus of the adenovirus type 2 genome and its use in site-directed mutagenesis. J Virol 37(1):171–180PubMedPubMedCentralGoogle Scholar
  71. 71.
    He TC, Zhou S, da Costa LT et al (1998) A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 95(5):2509–2514CrossRefGoogle Scholar
  72. 72.
    Chartier C, Degryse E, Gantzer M et al (1996) Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J Virol 70(7):4805–4810PubMedPubMedCentralGoogle Scholar
  73. 73.
    Wu C, Lei X, Wang J et al (2011) Generation of a replication-deficient recombinant human adenovirus type 35 vector using bacteria-mediated homologous recombination. J Virol Methods 177(1):55–63.  https://doi.org/10.1016/j.jviromet.2011.06.016CrossRefPubMedGoogle Scholar
  74. 74.
    Luo J, Deng ZL, Luo X et al (2007) A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc 2(5):1236–1247.  https://doi.org/10.1038/nprot.2007.135CrossRefPubMedGoogle Scholar
  75. 75.
    Ng P, Parks RJ, Cummings DT et al (1999) A high-efficiency Cre/loxP-based system for construction of adenoviral vectors. Hum Gene Ther 10(16):2667–2672.  https://doi.org/10.1089/10430349950016708CrossRefPubMedGoogle Scholar
  76. 76.
    Chen L, Anton M, Graham FL (1996) Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre. Somat Cell Mol Genet 22(6):477–488CrossRefGoogle Scholar
  77. 77.
    Mittereder N, March KL, Trapnell BC (1996) Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol 70(11):7498–7509PubMedPubMedCentralGoogle Scholar
  78. 78.
    Maizel JV Jr, White DO, Scharff MD (1968) The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36(1):115–125CrossRefGoogle Scholar
  79. 79.
    Rux JJ, Burnett RM (2007) Large-scale purification and crystallization of adenovirus hexon. In: Wold WSM, Tollefson AE (eds) Adenovirus methods and protocols, Ad proteins, RNA lifecycle, host interactions, and phylogenetics, vol 2. Humana Press, Totowa, NJ, pp 231–250.  https://doi.org/10.1007/978-1-59745-277-9_17CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Ekramy E. Sayedahmed
    • 1
  • Rashmi Kumari
    • 1
  • Suresh K. Mittal
    • 1
    Email author
  1. 1.Department of Comparative Pathobiology, Purdue Institute for Inflammation, Immunology, and Infectious Disease, College of Veterinary MedicinePurdue UniversityWest LafayetteUSA

Personalised recommendations