title: Identification and validation of a linear protective neutralizing epitope in the β-pore domain of alpha toxin.
abstract: The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric β-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2β2-2β3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the β-pore of S. aureus alpha toxin that share structural and functional homology to β-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing determinant in alpha toxin could facilitate the development of an epitope-focused vaccine against S. aureus.
authors: Division of Hematology-Oncology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, 48105, United States of America; VA Ann Arbor Healthcare System, 2215 Fuller Road, Ann Arbor, Michigan, 48105, United States of America.
authors: Jon
authors: J
authors: Oscherwitz
authors: Division of Hematology-Oncology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, 48105, United States of America; VA Ann Arbor Healthcare System, 2215 Fuller Road, Ann Arbor, Michigan, 48105, United States of America.
authors: Kemp B
authors: KB
authors: Cease
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000502:physiology; D057134:Antibodies, Neutralizing / Q000502:physiology; D000920:Antibody-Dependent Cell Cytotoxicity; D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology; D001427:Bacterial Toxins / Q000737:chemistry* / Q000276:immunology; D001428:Bacterial Vaccines / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D005098:Exotoxins / Q000737:chemistry* / Q000276:immunology; D005260:Female; D006801:Humans; D007116:Immunization, Passive; D019169:Jurkat Cells; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013203:Staphylococcal Infections / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control; D013207:Staphylococcal Skin Infections / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control; D013211:Staphylococcus aureus / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D057134:Antibodies, Neutralizing; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D000939:Epitopes; D005098:Exotoxins
references: Infect Immun. 1999 Mar;67(3):1045-9
references: 10024541
references: Clin Exp Immunol. 2003 May;132(2):209-15
references: 12699407
references: Clin Diagn Lab Immunol. 2003 Sep;10(5):903-9
references: 12965925
references: Vaccine. 2004 Jan 2;22(3-4):422-30
references: 14670324
references: Biologicals. 2004 Mar;32(1):17-27
references: 15026022
references: Clin Infect Dis. 2005 Feb 15;40(4):562-73
references: 15712079
references: Immunity. 2006 Jan;24(1):79-91
references: 16413925
references: Infect Immun. 2006 Dec;74(12):6929-39
references: 17030584
references: Infect Immun. 1991 Apr;59(4):1402-8
references: 1706321
references: Infect Immun. 2007 Feb;75(2):1040-4
references: 17101657
references: Int Immunol. 1991 Oct;3(10):997-1003
references: 1721837
references: BioDrugs. 2007;21(3):145-56
references: 17516710
references: Bioorg Med Chem. 2007 Aug 15;15(16):5424-31
references: 17572091
references: Microbiol Rev. 1991 Dec;55(4):733-51
references: 1779933
references: JAMA. 2007 Oct 17;298(15):1763-71
references: 17940231
references: Cell Mol Life Sci. 2008 Feb;65(3):493-507
references: 17989920
references: Nat Med. 2007 Dec;13(12):1510-4
references: 17994102
references: J Exp Med. 2008 Feb 18;205(2):287-94
references: 18268041
references: J Exp Med. 2008 Feb 18;205(2):271-4
references: 18268043
references: Vaccine. 2008 Jul 29;26(32):4041-7
references: 18565627
references: J Infect Dis. 2008 Nov 15;198(10):1529-35
references: 18823272
references: Infect Immun. 2009 May;77(5):2030-5
references: 19223482
references: Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5883-8
references: 19293374
references: Infect Immun. 2009 Jul;77(7):2712-8
references: 19380475
references: Infect Immun. 2009 Aug;77(8):3380-8
references: 19487468
references: J Immunol. 2009 Sep 15;183(6):3942-8
references: 19717510
references: Clin Vaccine Immunol. 2009 Dec;16(12):1709-19
references: 19793898
references: Infect Immun. 2009 Dec;77(12):5509-18
references: 19805525
references: Lancet. 2010 May 1;375(9725):1557-68
references: 20206987
references: J Clin Invest. 2010 May;120(5):1762-73
references: 20364087
references: Clin Microbiol Rev. 2010 Jul;23(3):616-87
references: 20610826
references: Proc Natl Acad Sci U S A. 2010 Jul 27;107(30):13473-8
references: 20624979
references: J Immunol. 2010 Sep 15;185(6):3661-8
references: 20696862
references: J Infect Dis. 2010 Oct 1;202(7):1050-8
references: 20726702
references: Annu Rev Microbiol. 2010;64:143-62
references: 20825344
references: Proc Natl Acad Sci U S A. 2010 Oct 19;107(42):17880-7
references: 20876137
references: J Infect Dis. 2010 Dec 15;202(12):1866-76
references: 21050125
references: Nat Med. 2011 Sep 18;17(10):1310-4
references: 21926978
references: Clin Infect Dis. 2012 Feb 15;54(4):560-7
references: 22186773
references: Clin Vaccine Immunol. 2012 Mar;19(3):377-85
references: 22237895
references: J Invest Dermatol. 2012 May;132(5):1513-6
references: 22377761
references: J Infect Dis. 2012 Aug 1;206(3):352-6
references: 22474035
references: PLoS One. 2012;7(5):e36532
references: 22574180
references: PLoS One. 2012;7(6):e38567
references: 22701668
references: Infect Immun. 2012 Oct;80(10):3721-32
references: 22802349
references: Nat Rev Microbiol. 2012 Dec;10(12):807-13
references: 23154260
references: Clin Vaccine Immunol. 2013 Mar;20(3):341-9
references: 23283638
references: ChemMedChem. 2013 Mar;8(3):360-76
references: 23316023
references: Infect Immun. 2013 Apr;81(4):1306-15
references: 23381997
references: Proc Natl Acad Sci U S A. 2013 Feb 26;110(9):3304-9
references: 23396847
references: J Mol Biol. 2013 May 27;425(10):1641-54
references: 23416200
references: PLoS One. 2013;8(3):e59232
references: 23555002
references: PLoS One. 2013 Apr 29;8(4):e63040
references: 23658662
references: Toxins (Basel). 2013 Jun;5(6):1140-66
references: 23888516
references: PLoS One. 2013 Oct 02;8(10):e75103
references: 24098366
references: Infect Immun. 2014 Jan;82(1):83-91
references: 24126523
references: PLoS One. 2013 Dec 04;8(12):e81212
references: 24324681
references: J Infect Dis. 2014 Jun 15;209(12):1955-62
references: 24357631
references: Nature. 2014 Mar 13;507(7491):201-6
references: 24499818
references: Clin Vaccine Immunol. 2014 May;21(5):622-7
references: 24574539
references: Mol Immunol. 2014 Jul;60(1):62-71
references: 24769493
references: Eur J Immunol. 1989 Dec;19(12):2237-42
references: 2481588
references: Proc Natl Acad Sci U S A. 1988 Aug;85(15):5409-13
references: 3399498
references: Microb Pathog. 1986 Apr;1(2):125-38
references: 3508485
references: Infect Immun. 1984 Nov;46(2):318-23
references: 6500692
references: J Biol Chem. 1995 Sep 29;270(39):23065-71
references: 7559447
references: Infect Immun. 1994 May;62(5):1843-7
references: 8168947
references: Infect Immun. 1996 May;64(5):1839-41
references: 8613399
references: Science. 1996 Dec 13;274(5294):1859-66
references: 8943190
references: J Immunol. 1997 Aug 1;159(3):1362-73
references: 9233633
references: JAMA. 1998 Feb 25;279(8):593-8
references: 9486753