2204. Human Monoclonal Antibodies to the Staphylococcus aureus Toxin LukAB have Distinct Mechanisms of Protection and Are Efficacious in vivo
Session: Poster Abstract Session: Host-Pathogen Interactions
Saturday, October 29, 2016
Room: Poster Hall

Background: Staphylococcus aureus (SA) is the most common invasive bacterial pathogen, and novel targets of intervention are urgently needed. The two-component leukotoxin, LukAB, is critical for SA targeting and killing of human neutrophils ex vivo and is produced in the setting of human infection. We report here three LukAB-specific human monoclonal antibodies (mAb) with distinct mechanisms of toxin neutralization and in vivo efficacy.

Methods: B-cells were isolated from children with invasive SA infections. Following EBV-transformation, cell supernatants were screened for LukAB binding and neutralization. Cells from positive wells were fused with myeloma cells and cultured in selection medium for generation of monoclonal hybridomas. MAbs were assessed for binding by ELISA and neutralizing function by in vitro cytotoxicity assays and ex in vivo infection assays. C57BL/6 mice were dosed with mAbs one day prior to initiation of SA sepsis via retroorbital injection, and tissue colony counts were obtained on day 4 post-infection.

Results: Three hybridomas secreting mAbs with anti-LukAB activity (SA-13, -15, and -17) were generated from a 12 year-old boy with SA osteomyelitis. All three mAbs neutralized LukAB-mediated cytotoxicity with diverse potency in vitro (Fig. 1). SA-17 had a distinct mechanism of neutralization, inhibiting toxin association with the cell surface (Fig. 2). Both SA-13 and SA-17 bound the LukA monomer and the LukAB dimer; however, SA-15 bound only the LukAB dimer, indicating a binding site formed only after dimerization. A 1:1 mixture of SA-15 and SA-17 resulted in significantly lower colony counts in murine heart, liver, and kidneys following SA sepsis (Fig. 3).

Conclusion: MAbs purified following human SA infection potently neutralized LukAB-mediated cytotoxicity. While all three mAbs neutralized LukAB, they exhibited differing levels of potency, recognize different antigenic sites, and used distinct mechanisms of toxin inhibition. SA-15 exclusively bound the dimeric form of the toxin, suggesting that human B cells do recognize the dimerized form of LukAB during natural infection. These represent the first known broadly active human mAbs against LukAB, a toxin with high potential as a novel vaccine or therapeutic target.


Isaac Thomsen, MD, MSCI1, David James, PhD2, James Cassat, MD, PhD1, Gopal Sapparapu, PhD3, C Creech, MD, MPH, FPIDS1, Victor Torres, Ph.D.4 and James E. Crowe Jr., MD, FIDSA5, (1)Pediatric Infectious Diseases, Vanderbilt University Medical Center, Nashville, TN, (2)Microbiology, New York University, New York, NY, (3)Vanderbilt University Medical Center, Nashville, TN, (4)Department of Microbiology, New York University School of Medicine, New York, NY, (5)Pediatrics and Microbiology and Immunology, Vanderbilt University, Nashville, TN

Disclosures:

I. Thomsen, None

D. James, None

J. Cassat, None

G. Sapparapu, None

C. Creech, None

V. Torres, None

J. E. Crowe Jr., None

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