Characterization of Alpha Toxin hla Gene Variants, Alpha Toxin

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    Characterization of Alpha Toxin hla Gene Variants, Alpha Toxin Expression 1

    Levels and Antibody Levels to Alpha Toxin in Hemodialysis and Post-Surgical 2

    Patients with Staphylococcus aureus Bacteremia 3

    4

    Running Title: -Toxin and antibodies in high-risk SAB patients 5

    6

    Batu K. Sharma-Kuinkel1*

    , Yuling Wu2, David E. Tabor

    2, Hoyin Mok

    2, Bret R. Sellman

    2, Amy 7

    Jenkins2, Li Yu

    2, Hasan S. Jafri

    2, Thomas H. Rude,

    1 Felicia Ruffin

    1, Wiley A. Schell

    1 , 8

    Lawrence P. Park1, Qin Yan

    1, Joshua T. Thaden

    1, Julia A. Messina

    1, Vance G. Fowler, Jr.

    1,3* and 9

    Mark T. Esser2

    10

    1Department of Medicine, Duke University Medical Center, Durham, North Carolina, United 11

    States of America 12

    2 MedImmune LLC., Gaithersburg, Maryland, United States of America 13

    3Duke Clinical Research Institute, Durham, North Carolina, United States of America 14

    *Contact information: 15

    Vance G. Fowler Jr., MD, MHS 16

    Division of Infectious Diseases 17

    Department of Medicine, 18

    Duke University Medical Center 19

    Box 102359 Medical Center, Durham, NC 27710 20

    Tel: (919) 613-5678 21

    Fax: (919) 684-8902 22

    *E-mail: [email protected] 23

    Batu K. Sharma-Kuinkel 24

    JCM Accepts, published online ahead of print on 12 November 2014J. Clin. Microbiol. doi:10.1128/JCM.02023-14Copyright 2014, American Society for Microbiology. All Rights Reserved.

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    *E-mail: [email protected] 25

    26

    ABSTRACT 27

    Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated 28

    potential relationships between in vitro -toxin expression of S. aureus bloodstream isolates, 29

    anti--toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcome 30

    in 100 hemodialysis and 100 post-surgical SAB patients. Isolates underwent spa-typing and hla 31

    sequencing. Serum anti--toxin IgG and neutralizing antibody levels were measured using 32

    ELISA and RBC-based hemolysis neutralization assay. Neutralization of -toxin by anti--toxin 33

    mAb (MEDI4893) was tested in an RBCbased lysis assay. Most isolates encoded hla (197/200; 34

    98.5%) and expressed -toxin (173/200; 86.5%). In vitro -toxin levels were inversely 35

    associated with survival (Cure: 2.19 g/ml vs. Failure: 1.09 g/ml; P < 0.01). Both neutralizing 36

    (Hemodialysis: 1.26 IU/ml vs. Post-surgical: 0.95; P

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    toxin antibody levels, these levels were not associated with a better clinical outcome in this 46

    study. 47

    48

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    INTRODUCTION 49 Staphylococcus aureus is a leading cause of bacterial infections(1-4) including skin and 50

    soft tissue (5), pneumonia (6), bacteremia (7), endocarditis (8-10), and bone and joints (11). The 51

    risk of invasive S. aureus infections is significantly higher among certain subgroups including 52

    hemodialysis-dependent patients and postoperative patients (12-14).These high risk 53

    subpopulations are potential candidates for novel forms of prevention or treatment against 54

    invasive S. aureus infections. 55

    Alpha-toxin, a beta-barrel pore-forming exotoxin encoded by hla (15, 16), is a key 56

    virulence factor produced by most S. aureus isolates (17, 18). It binds to ADAM 10 (A 57

    disintegrin and metalloproteinase domain-containing protein 10) on target cell membranes and 58

    then heptamerizes to generate a transmembrane pore resulting in cell lysis (19). Hyper-59

    production of -toxin is associated with enhanced virulence in strains of both epidemic 60

    (USA300, USA500) and endemic (ST93) community-associated methicillin-resistant S. aureus 61

    (CA-MRSA) isolates (20, 21). A number of animal models also suggest that -toxin is a key 62

    virulence factor in the pathogenesis of S. aureus infections including pneumonia (22-25), skin 63

    and soft tissue infections (26, 27) and bloodstream infections (28). 64

    Alpha toxin represents an attractive target for novel immunotherapeutic approaches of 65

    prevention or treatment of invasive S. aureus infections (29-31). In murine S. aureus skin and 66

    soft tissue infection models, both active immunization with a non-toxigenic -toxin mutant and 67

    passive immunization with -toxin-specific antisera or IgG significantly improved survival and 68

    reduced disease severity (27, 32-34). Antibodies against -toxin were both necessary and 69

    sufficient for protection against soft tissue infection and were inversely associated with bacterial 70

    counts (32) and had a protective role in keratitis (35). Also, a recent report suggests that higher 71

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    antibody levels against -toxin have a protective role against sepsis in patients with invasive S. 72

    aureus infections (36). Despite these important insights, the complex interplay between -toxin 73

    production by bloodstream S. aureus isolates, anti- -toxin antibody production by bacteremic 74

    patients, and the clinical outcome of those patients is incompletely understood. 75

    In the current study, we assessed in vitro -toxin expression in S. aureus isolates and anti-76

    -toxin antibodies in patients with S. aureus bacteremia (SAB). Using acute phase sera and the 77

    associated bloodstream bacterial isolates from a cohort of prospectively ascertained, clinically 78

    well-characterized patients with SAB, we evaluated potential relationships between in vitro -79

    toxin production and anti--toxin-specific IgG and neutralizing antibodies levels to clinical 80

    outcome. We also sequenced hla from the bloodstream isolates to explore relationships between 81

    variation of hla and the clinical severity of infection. Finally, we tested the effectiveness of 82

    MEDI4893, a broadly reactive anti--toxin mAb, to neutralize -toxin expressed by all of the hla 83

    variants identified in this investigation. 84

    MATERIALS AND METHODS 85

    Patient selection and ethics statement 86

    The Duke University Institutional Review Board approved this investigation. Eligible 87

    patients for the current study met all of the following criteria: (1) Unique adult patients 88

    hospitalized at Duke University Medical Center with mono-microbial S. aureus bacteremia; (2) 89

    No neutropenia (Absolute neutrophil count > 100 neutrophils/l); (3) Availability of a) clinical 90

    data; b) bloodstream S. aureus isolate and c) acute phase sera in the Staphylococcus aureus 91

    Bacteremia Group (SABG) Biorepository; and (4) Presence of either a) end-stage renal disease 92

    necessitating chronic hemodialysis; or b) surgical procedure in the preceding 30 days. Samples 93

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    from 100 hemodialysis patients and 100 post-surgical patients meeting these criteria and 94

    collected from July 2001 to December 2009 were included in this study. Serum samples from 95

    healthy adults age 18-60 years old were purchased from Bioreclamation, Westbury, NY and used 96

    as controls. 97

    Clinical Outcomes and Definitions 98

    Clinical outcomes were established by site investigators. Outcomes were defined as 99

    either Cure or Failure. A patients outcome was defined as a Cure if he or she was alive 100

    with no evidence of recurrent S. aureus infection at 12 weeks following the initial positive blood 101

    culture. An outcome of Failure was defined if a patient died for any reason or experienced 102

    culture-confirmed recurrent S. aureus infection within 12 weeks of the initial positive blood 103

    culture. Hemodialysis patients were defined as the patients with end-stage renal disease 104

    necessitating chronic hemodialysis. Post-surgical patients were defined as the patients who had 105

    undergone surgical procedure in the preceding 30 days. 106

    Staphylococcus aureus isolates 107

    S. aureus isolates were identified from the blood stream of patients providing written 108

    informed consent and were stored at -80C until ready for use. Isolates were linked to the 109

    clinical details relevant to their case by way of a unique study number. For patients with multiple 110

    positive blood cultures, the initial isolate was used for all characterizations. 111

    Spa typing and identification of clonal complex 112

    S. aureus genomic DNA was extracted as described previously (37), using an ultraclean 113

    microbial DNA kit (MO BIO Laboratories, Inc., Carlsbad, CA) in accordance with the 114

    manufacturers instructions. Spa typing was performed as previously described (38, 39). Briefly, 115

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    eGenomics software (http://tools.egenomics.com/) was used to scan the primary sequences and 116

    to determine the spa types. The spa type number is representative of the repeat organization. 117

    Clona