Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection

PLoS Pathogens

Volumn 11, Issue 11, 2015, Pages

  Sommerstein, Rami ^a,h   Flatz, Lukas ^a,i   Remy, Melissa M ^a,b   Malinge, Pauline ^c   Magistrelli, Giovanni ^c   Fischer, Nicolas ^c   Sahin, Mehmet ^b   Bergthaler, Andreas ^a,j   Igonet, Sebastien ^d,k   ter Meulen, Jan ^e,l   Rigo, Dorothée ^a   Meda, Paolo ^a   Rabah, Nadia ^f   Coutard, Bruno ^f   Bowden, Thomas A ^g   Lambert, Paul Henri ^a   Siegrist, Claire Anne ^a   Pinschewer, Daniel D ^a,b  

^a UNIVERSITY OF GENEVA   (Switzerland)

^b UNIVERSITY OF BASEL   (Switzerland)

^c NOVIMMUNE SA   (Switzerland)

^d INSTITUT PASTEUR   (France)

^e PHILIPPS UNIVERSITY MARBURG   (Germany)

^f AIX MARSEILLE UNIVERSITY   (France)

^g UNIVERSITY OF OXFORD   (United Kingdom)

^h UNIVERSITY OF BERN   (Switzerland)

ⁱ Institute of Immunobiology and Clinic for Dermatology and Allergology   (Switzerland)

^j CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences ^*  (Austria)

^k Calixar SAS   (France)

^l Immune Design   (United States)

more..

Author keywords

[No Author keywords available]

Indexed keywords

GLYCAN; NEUTRALIZING ANTIBODY; HUMAN IMMUNODEFICIENCY VIRUS ANTIBODY; POLYSACCHARIDE; VIRUS ANTIBODY;

ANTIBODY RESPONSE; ANTIGEN BINDING; ARTICLE; CONTROLLED STUDY; ELECTRON MICROSCOPY; ENZYME LINKED IMMUNOSORBENT ASSAY; FLOW CYTOMETRY; HUMAN; IMMUNE EVASION; LASSA VIRUS; MOUSE; NONHUMAN; PROTEIN EXPRESSION; PROTEIN GLYCOSYLATION; SEQUENCE ALIGNMENT; SURFACE PLASMON RESONANCE; VIRUS INFECTION; VIRUS TITRATION; ANIMAL; ARENAVIRUS; C57BL MOUSE; HUMAN IMMUNODEFICIENCY VIRUS 1; IMMUNOLOGY; MOLECULAR GENETICS; VIRUS HEMORRHAGIC FEVER;

ANIMALS; ANTIBODIES, NEUTRALIZING; ANTIBODIES, VIRAL; ARENAVIRUS; HEMORRHAGIC FEVERS, VIRAL; HIV ANTIBODIES; HIV-1; HUMANS; MICE, INBRED C57BL; MOLECULAR SEQUENCE DATA; POLYSACCHARIDES;

EID: 84948964373     PISSN: 15537366     EISSN: 15537374     Source Type: Journal    
DOI: 10.1371/journal.ppat.1005276     Document Type: Article

References (81)

1. Plotkin SA, Correlates of protection induced by vaccination. Clin Vaccine Immunol. 2010;17(7):1055–65. doi: CVI.00131-10 [pii] doi: 10.1128/CVI.00131-10 20463105.
2. Amanna IJ, Slifka MK, Contributions of humoral and cellular immunity to vaccine-induced protection in humans. Virology. 2011;411(2):206–15. doi: 10.1016/j.virol.2010.12.016 21216425; PubMed Central PMCID: PMC3238379.
3. Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, et al. Antibody neutralization and escape by HIV-1. Nature. 2003;422(6929):307–12. 12646921.
4. Helle F, Vieyres G, Elkrief L, Popescu CI, Wychowski C, Descamps V, et al. Role of N-linked glycans in the functions of hepatitis C virus envelope proteins incorporated into infectious virions. J Virol. 2010;84(22):11905–15. doi: 10.1128/JVI.01548-10 20844034; PubMed Central PMCID: PMC2977866.
5. Das SR, Puigbo P, Hensley SE, Hurt DE, Bennink JR, Yewdell JW, Glycosylation focuses sequence variation in the influenza A virus H1 hemagglutinin globular domain. PLoS Pathog. 2010;6(11):e1001211. doi: 10.1371/journal.ppat.1001211 21124818; PubMed Central PMCID: PMC2991263.
6. Fafi-Kremer S, Fofana I, Soulier E, Carolla P, Meuleman P, Leroux-Roels G, et al. Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation. J Exp Med. 2010;207(9):2019–31. doi: 10.1084/jem.20090766 20713596; PubMed Central PMCID: PMC2931157.
7. Richman DD, Wrin T, Little SJ, Petropoulos CJ, Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc Natl Acad Sci U S A. 2003;100(7):4144–9. 12644702.
8. Zlateva KT, Lemey P, Vandamme AM, Van Ranst M, Molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment g glycoprotein. J Virol. 2004;78(9):4675–83. 15078950; PubMed Central PMCID: PMC387670.
9. Charrel RN, de Lamballerie X, Emonet S, Phylogeny of the genus Arenavirus. Current opinion in microbiology. 2008;11(4):362–8. doi: 10.1016/j.mib.2008.06.001 18602020.
10. McCormick JB, Webb PA, Krebs JW, Johnson KM, Smith ES, A prospective study of the epidemiology and ecology of Lassa fever. J Infect Dis. 1987;155(3):437–44. 3805771.
11. Maiztegui JI, McKee KT, Jr.Barrera Oro JG, Harrison LH, Gibbs PH, Feuillade MR, et al. Protective efficacy of a live attenuated vaccine against Argentine hemorrhagic fever. AHF Study Group. J Infect Dis. 1998;177(2):277–83. 9466512.
12. Olschlager S, Flatz L, Vaccination strategies against highly pathogenic arenaviruses: the next steps toward clinical trials. PLoS Pathog. 2013;9(4):e1003212. doi: 10.1371/journal.ppat.1003212 23592977; PubMed Central PMCID: PMC3623805.
13. McCormick JB, King IJ, Webb PA, Scribner CL, Craven RB, Johnson KM, et al. Lassa fever. Effective therapy with ribavirin. N Engl J Med. 1986;314(1):20–6. 3940312.
14. Casals J, Buckley SM, Cedeno R, Antigenic properties of the arenaviruses. Bulletin of the World Health Organization. 1975;52(4–6):421–7. 182397; PubMed Central PMCID: PMC2366642.
15. Jahrling PB, Frame JD, Rhoderick JB, Monson MH, Endemic Lassa fever in Liberia. IV. Selection of optimally effective plasma for treatment by passive immunization. Trans R Soc Trop Med Hyg. 1985;79(3):380–4. 3898484.
16. Jahrling PB, Peters CJ, Passive antibody therapy of Lassa fever in cynomolgus monkeys: importance of neutralizing antibody and Lassa virus strain. Infect Immun. 1984;44(2):528–33. 6715049.
17. Clayton AJ, Lassa immune serum. Bulletin of the World Health Organization. 1977;55(4):435–9. 304386; PubMed Central PMCID: PMC2366682.
18. McCormick JB, Mitchell SW, Kiley MP, Ruo S, Fisher-Hoch SP, Inactivated Lassa virus elicits a non protective immune response in rhesus monkeys. J Med Virol. 1992;37(1):1–7. 1619397.
19. Walker DH, McCormick JB, Johnson KM, Webb PA, Komba-Kono G, Elliott LH, et al. Pathologic and virologic study of fatal Lassa fever in man. Am J Pathol. 1982;107(3):349–56. 7081389
20. Gonzalez PH, Cossio PM, Arana R, Maiztegui JI, Laguens RP, Lymphatic tissue in Argentine hemorrhagic fever. Pathologic features. Archives of pathology & laboratory medicine. 1980;104(5):250–4. 6154445.
21. Enria DA, Briggiler AM, Fernandez NJ, Levis SC, Maiztegui JI, Importance of dose of neutralising antibodies in treatment of Argentine haemorrhagic fever with immune plasma. Lancet. 1984;2(8397):255–6. 6146809.
22. Videla C, Carballal G, Remorini P, La Torre J, Formalin inactivated Junin virus: immunogenicity and protection assays. J Med Virol. 1989;29(3):215–20. 2559158.
23. Buchmeier MJ, Arenaviruses: Protein Structure and Function. in Curr Top Microb Immunol. 2002;262:159–73.
24. Battegay M, Moskophidis D, Waldner H, Brundler MA, Fung-Leung WP, Mak TW, et al. Impairment and delay of neutralizing antiviral antibody responses by virus-specific cytotoxic T cells. J Immunol. 1993;151(10):5408–15. 7693811.
25. Eschli B, Zellweger RM, Wepf A, Lang KS, Quirin K, Weber J, et al. Early antibodies specific for the neutralizing epitope on the receptor binding subunit of the lymphocytic choriomeningitis virus glycoprotein fail to neutralize the virus. J Virol. 2007;81(21):11650–7. doi: JVI.00955-07 [pii] doi: 10.1128/JVI.00955-07 17699567.
26. Bergthaler A, Flatz L, Verschoor A, Hegazy AN, Holdener M, Fink K, et al. Impaired antibody response causes persistence of prototypic T cell-contained virus. PLoS Biol. 2009;7(4):e1000080. doi: 08-PLBI-RA-3478 [pii] doi: 10.1371/journal.pbio.1000080 19355789.
27. Pinschewer DD, Perez M, Jeetendra E, Bächi T, Horvath E, Hengartner H, et al. Kinetics of protective antibodies are determined by the viral surface antigen. J Clin Invest. 2004;114:988–93. 15467838
28. Bruns M, Cihak J, Muller G, Lehmann-Grube F, Lymphocytic choriomeningitis virus. VI. Isolation of a glycoprotein mediating neutralization. Virology. 1983;130(1):247–51. 6636539.
29. Seiler P, Brundler MA, Zimmermann C, Weibel D, Bruns M, Hengartner H, et al. Induction of protective cytotoxic T cell responses in the presence of high titers of virus-neutralizing antibodies: implications for passive and active immunization. J Exp Med. 1998;187(4):649–54. 9463415.
30. Andersen KG, Shapiro BJ, Matranga CB, Sealfon R, Lin AE, Moses LM, et al. Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus. Cell. 2015;162(4):738–50. doi: 10.1016/j.cell.2015.07.020 26276630; PubMed Central PMCID: PMC4537774.
31. Eichler R, Lenz O, Garten W, Strecker T, The role of single N-glycans in proteolytic processing and cell surface transport of the Lassa virus glycoprotein GP-C. Virol J. 2006;3:41. doi: 10.1186/1743-422X-3-41 16737539; PubMed Central PMCID: PMC1524727.
32. Bonhomme CJ, Capul AA, Lauron EJ, Bederka LH, Knopp KA, Buchmeier MJ, Glycosylation modulates arenavirus glycoprotein expression and function. Virology. 2011;409(2):223–33. doi: 10.1016/j.virol.2010.10.011 21056893; PubMed Central PMCID: PMC3053032.
33. Helenius A, Aebi M, Intracellular functions of N-linked glycans. Science. 2001;291(5512):2364–9. 11269317.
34. Wright KE, Salvato MS, Buchmeier MJ, Neutralizing epitopes of lymphocytic choriomeningitis virus are conformational and require both glycosylation and disulfide bonds for expression. Virology. 1989;171(2):417–26. 2474891.
35. Bonhomme CJ, Knopp KA, Bederka LH, Angelini MM, Buchmeier MJ, LCMV glycosylation modulates viral fitness and cell tropism. PLoS One. 2013;8(1):e53273. doi: 10.1371/journal.pone.0053273 23308183; PubMed Central PMCID: PMC3538765.
36. Schwarz F, Aebi M, Mechanisms and principles of N-linked protein glycosylation. Current opinion in structural biology. 2011;21(5):576–82. doi: 10.1016/j.sbi.2011.08.005 21978957.
37. Trapido H, Sanmartin C, Pichinde virus, a new virus of the Tacaribe group from Colombia. Am J Trop Med Hyg. 1971;20(4):631–41. 4998616.
38. Charrel RN, de Lamballerie X, Arenaviruses other than Lassa virus. Antiviral Res. 2003;57(1–2):89–100. 12615305.
39. Albarino CG, Bird BH, Chakrabarti AK, Dodd KA, Flint M, Bergeron E, et al. The major determinant of attenuation in mice of the Candid1 vaccine for Argentine hemorrhagic fever is located in the G2 glycoprotein transmembrane domain. J Virol. 2011;85(19):10404–8. doi: 10.1128/JVI.00856-11 21795336; PubMed Central PMCID: PMC3196416.
40. McKee KT, Jr.Oro JG, Kuehne AI, Spisso JA, Mahlandt BG, Candid No. 1 Argentine hemorrhagic fever vaccine protects against lethal Junin virus challenge in rhesus macaques. Intervirology. 1992;34(3):154–63. 1338783.
41. Grant A, Seregin A, Huang C, Kolokoltsova O, Brasier A, Peters C, et al. Junin virus pathogenesis and virus replication. Viruses. 2012;4(10):2317–39. doi: 10.3390/v4102317 23202466; PubMed Central PMCID: PMC3497054.
42. ter Meulen J, Badusche M, Kuhnt K, Doetze A, Satoguina J, Marti T, et al. Characterization of human CD4(+) T-cell clones recognizing conserved and variable epitopes of the Lassa virus nucleoprotein. J Virol. 2000;74(5):2186–92. 10666248.
43. Seiler P, Senn BM, Brundler MA, Zinkernagel RM, Hengartner H, Kalinke U, In vivo selection of neutralization-resistant virus variants but no evidence of B cell tolerance in lymphocytic choriomeningitis virus carrier mice expressing a transgenic virus-neutralizing antibody. J Immunol. 1999;162(8):4536–41. 10201992.
44. Hangartner L, Zellweger RM, Giobbi M, Weber J, Eschli B, McCoy KD, et al. Nonneutralizing antibodies binding to the surface glycoprotein of lymphocytic choriomeningitis virus reduce early virus spread. J Exp Med. 2006;203(8):2033–42. doi: jem.20051557 [pii] doi: 10.1084/jem.20051557 16880253.
45. Sanchez A, Pifat DY, Kenyon RH, Peters CJ, McCormick JB, Kiley MP, Junin virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses. J Gen Virol. 1989;70 (Pt 5):1125–32. 2471803.
46. Wu H, Pfarr DS, Tang Y, An LL, Patel NK, Watkins JD, et al. Ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization. J Mol Biol. 2005;350(1):126–44. doi: 10.1016/j.jmb.2005.04.049 15907931.
47. McKinley SA, Chen A, Shi F, Wang S, Mucha PJ, Forest MG, et al. Modeling neutralization kinetics of HIV by broadly neutralizing monoclonal antibodies in genital secretions coating the cervicovaginal mucosa. PLoS One. 2014;9(6):e100598. doi: 10.1371/journal.pone.0100598 24967706; PubMed Central PMCID: PMC4072659.
48. Bowden TA, Crispin M, Graham SC, Harvey DJ, Grimes JM, Jones EY, et al. Unusual molecular architecture of the machupo virus attachment glycoprotein. J Virol. 2009;83(16):8259–65. doi: 10.1128/JVI.00761-09 19494008; PubMed Central PMCID: PMC2715760.
49. Abraham J, Corbett KD, Farzan M, Choe H, Harrison SC, Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses. Nature structural & molecular biology. 2010;17(4):438–44. doi: 10.1038/nsmb.1772 20208545; PubMed Central PMCID: PMC2920743.
50. Kelley LA, Sternberg MJ, Protein structure prediction on the Web: a case study using the Phyre server. Nature protocols. 2009;4(3):363–71. doi: 10.1038/nprot.2009.2 19247286.
51. Radoshitzky SR, Abraham J, Spiropoulou CF, Kuhn JH, Nguyen D, Li W, et al. Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses. Nature. 2007;446(7131):92–6. doi: 10.1038/nature05539 17287727; PubMed Central PMCID: PMC3197705.
52. Cao W, Henry MD, Borrow P, Yamada H, Elder JH, Ravkov EV, et al. Identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus. Science. 1998;282(5396):2079–81. 9851928.
53. Sanna PP, Burton DR, Role of antibodies in controlling viral disease: lessons from experiments of nature and gene knockouts. J Virol. 2000;74(21):9813–7. 11024107.
54. Ahmed R, Salmi A, Butler LD, Chiller JM, Oldstone MB, Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice. Role in suppression of cytotoxic T lymphocyte response and viral persistence. J Exp Med. 1984;160(2):521–40. 6332167.
55. Penaloza-MacMaster P, Barber DL, Wherry EJ, Provine NM, Teigler JE, Parenteau L, et al. Vaccine-elicited CD4 T cells induce immunopathology after chronic LCMV infection. Science. 2015;347(6219):278–82. doi: 10.1126/science.aaa2148 25593185; PubMed Central PMCID: PMC4382081.
56. Fisher-Hoch SP, Hutwagner L, Brown B, McCormick JB, Effective vaccine for lassa fever. J Virol. 2000;74(15):6777–83. 10888616.
57. Geisbert TW, Jones S, Fritz EA, Shurtleff AC, Geisbert JB, Liebscher R, et al. Development of a new vaccine for the prevention of Lassa fever. PLoS Med. 2005;2(6):e183. 15971954.
58. Lukashevich IS, Carrion R, Jr.Salvato MS, Mansfield K, Brasky K, Zapata J, et al. Safety, immunogenicity, and efficacy of the ML29 reassortant vaccine for Lassa fever in small non-human primates. Vaccine. 2008;26(41):5246–54. doi: 10.1016/j.vaccine.2008.07.057 18692539; PubMed Central PMCID: PMC2582173.
59. Kiley MP, Lange JV, Johnson KM, Protection of rhesus monkeys from Lassa virus by immunisation with closely related Arenavirus. Lancet. 1979;2(8145):738. 90819.
60. Wyatt R, Kwong PD, Desjardins E, Sweet RW, Robinson J, Hendrickson WA, et al. The antigenic structure of the HIV gp120 envelope glycoprotein. Nature. 1998;393(6686):705–11. doi: 10.1038/31514 9641684.
61. Munro JB, Gorman J, Ma X, Zhou Z, Arthos J, Burton DR, et al. Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions. Science. 2014. doi: 10.1126/science.1254426 25298114.
62. Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, et al. Emergence of Zaire Ebola virus disease in Guinea. N Engl J Med. 2014;371(15):1418–25. doi: 10.1056/NEJMoa1404505 24738640.
63. Lukashevich IS, Advanced vaccine candidates for Lassa fever. Viruses. 2012;4(11):2514–57. doi: 10.3390/v4112514 23202493; PubMed Central PMCID: PMC3509661.
64. Flatz L, Rieger T, Merkler D, Bergthaler A, Regen T, Schedensack M, et al. T cell-dependence of Lassa fever pathogenesis. PLoS Pathog. 2010;6(3):e1000836. doi: 10.1371/journal.ppat.1000836 20360949.
65. Peters CJ, Jahrling PB, Liu CT, Kenyon RH, McKee KT, Jr.Barrera Oro JG, Experimental studies of arenaviral hemorrhagic fevers. Curr Top Microbiol Immunol. 1987;134:5–68. 3034512.
66. Huttner A, Dayer JA, Yerly S, Combescure C, Auderset F, Desmeules J, et al. The effect of dose on the safety and immunogenicity of the VSV Ebola candidate vaccine: a randomised double-blind, placebo-controlled phase 1/2 trial. Lancet Infect Dis. 2015. doi: 10.1016/S1473-3099(15)00154-1 26248510.
67. Agnandji ST, Huttner A, Zinser ME, Njuguna P, Dahlke C, Fernandes JF, et al. Phase 1 Trials of rVSV Ebola Vaccine in Africa and Europe—Preliminary Report. N Engl J Med. 2015. doi: 10.1056/NEJMoa1502924 25830326.
68. Correia BE, Bates JT, Loomis RJ, Baneyx G, Carrico C, Jardine JG, et al. Proof of principle for epitope-focused vaccine design. Nature. 2014;507(7491):201–6. doi: 10.1038/nature12966 24499818.
69. Zhou T, Zhu J, Yang Y, Gorman J, Ofek G, Srivatsan S, et al. Transplanting supersites of HIV-1 vulnerability. PLoS One. 2014;9(7):e99881. doi: 10.1371/journal.pone.0099881 24992528; PubMed Central PMCID: PMC4084637.
70. Seregin AV, Yun NE, Miller M, Aronson J, Smith JK, Walker AG, et al. The glycoprotein precursor gene of Junin virus determines the virulence of the Romero strain and the attenuation of the Candid #1 strain in a representative animal model of Argentine hemorrhagic fever. J Virol. 2015;89(11):5949–56. doi: 10.1128/JVI.00104-15 25810546; PubMed Central PMCID: PMC4442433.
71. Barrios C, Brawand P, Berney M, Brandt C, Lambert PH, Siegrist CA, Neonatal and early life immune responses to various forms of vaccine antigens qualitatively differ from adult responses: predominance of a Th2-biased pattern which persists after adult boosting. Eur J Immunol. 1996;26(7):1489–96. doi: 10.1002/eji.1830260713 8766551.
72. Campetella OE, Galassi NV, Sanjuan N, Barrios HA, Susceptible adult murine model for Junin virus. J Med Virol. 1988;26(4):443–51. 2850346.
73. Flatz L, Bergthaler A, de la Torre JC, Pinschewer DD, Recovery of an arenavirus entirely from RNA polymerase I/II-driven cDNA. Proc Natl Acad Sci U S A. 2006;103(12):4663–8. 16537369.
74. Pinschewer DD, Perez M, Sanchez AB, de la Torre JC, Recombinant lymphocytic choriomeningitis virus expressing vesicular stomatitis virus glycoprotein. Proc Natl Acad Sci U S A. 2003;100(13):7895–900. 12808132.
75. Flatz L, Hegazy AN, Bergthaler A, Verschoor A, Claus C, Fernandez M, et al. Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8(+) T cell immunity. Nat Med. 2010;16(3):339–45. doi: nm.2104 [pii] doi: 10.1038/nm.2104 20139992.
76. Battegay M, Cooper S, Althage A, Banziger J, Hengartner H, Zinkernagel RM, Quantification of lymphocytic choriomeningitis virus with an immunological focus assay in 24- or 96-well plates. J Virol Methods. 1991;33(1–2):191–8. 1939506.
77. Geuze JJ, Slot JW, Tokuyasu KT, Immunocytochemical localization of amylase and chymotrypsinogen in the exocrine pancreatic cell with special attention to the Golgi complex. The Journal of cell biology. 1979;82(3):697–707. 511932; PubMed Central PMCID: PMC2110482.
78. Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ, Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25(9):1189–91. doi: 10.1093/bioinformatics/btp033 19151095; PubMed Central PMCID: PMC2672624.
79. Combet C, Blanchet C, Geourjon C, Deleage G, NPS@: network protein sequence analysis. Trends in biochemical sciences. 2000;25(3):147–50. 10694887.
80. Krissinel E, Henrick K, Inference of macromolecular assemblies from crystalline state. J Mol Biol. 2007;372(3):774–97. doi: 10.1016/j.jmb.2007.05.022 17681537.
81. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389–402. 9254694; PubMed Central PMCID: PMC146917.