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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: fowle003@mc.duke.edu 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: batu.sharma@duke.edu 25

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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

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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