Role of antibodies in protection elicited by active vaccination with genetically inactivated alpha hemolysin in a mouse model of staphylococcus aureus skin and soft tissue infections.

Clin Vaccine Immunol. 2014 May; 21(5): 622–627. doi: 10.1128/CVI.00051-14 PMCID: PMC4018873 PMID: 24574539 Role of Antibodies in Protection Elicited by Active Vaccination with Genetically Inactivated Alpha Hemolysin in a Mouse Model of Staphylococcus aureus Skin and Soft Tissue Infections Christopher P. Mocca, Rebecca A. Brady, and Drusilla L. Burns M. F. Pasetti, Editor Author information Article notes Copyright and License information Disclaimer Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA Corresponding author. Address correspondence to Drusilla L. Burns, . Received 2014 Jan 28; Revisions requested 2014 Feb 16; Accepted 2014 Feb 20. Copyright © 2014, American Society for Microbiology. All Rights Reserved. This article has been cited by other articles in PMC. Associated DataSupplementary Materials Supplemental material supp_21_5_622__index.html (967 bytes) GUID: 3C2D4CF3-397B-42B7-A16D-3E24D2B51147 CVI.00051-14_zcd999094963so1.pdf (31K) GUID: 8D319DA9-C51D-4145-9018-AD8D2086AF4B Abstract Due to the emergence of highly virulent community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, S. aureus has become a major threat to public health. A majority of CA-MRSA skin and soft tissue infections in the United States are caused by S. aureus USA300 strains that are known to produce high levels of alpha hemolysin (Hla). Therefore, vaccines that contain inactivated forms of this toxin are currently being developed. In this study, we sought to determine the immune mechanisms of protection for this antigen using a vaccine composed of a genetically inactivated form of Hla (HlaH35L). Using a murine model of skin and soft tissue infections (SSTI), we found that BALB/c mice were protected by vaccination with HlaH35L; however, Jh mice, which are deficient in mature B lymphocytes and lack IgM and IgG in their serum, were not protected. Passive immunization with anti-HlaH35L antibodies conferred protection against bacterial colonization. Moreover, we found a positive correlation between the total antibody concentration induced by active vaccination and reduced bacterial levels. Animals that developed detectable neutralizing antibody titers after active vaccination were significantly protected from infection. These data demonstrate that antibodies to Hla represent the major mechanism of protection afforded by active vaccination with inactivated Hla in this murine model of SSTI, and in this disease model, antibody levels correlate with protection. These results provide important information for the future development and evaluation of S. aureus vaccines. INTRODUCTION Staphylococcus aureus is an opportunistic pathogen that colonizes 30% of the population (1). This bacterium has a plethora of virulence factors that it uses to evade the immune system, scavenge for needed nutrients, and attach to and destroy host tissue (2, 3). S. aureus is a common cause of uncomplicated skin infections (4) but can also cause more invasive infections, like sepsis, endocarditis, and osteomyelitis (2). Methicillin-resistant S. aureus (MRSA), which is resistant to all β-lactam antibiotics, has historically been associated with nosocomial infections; however, community-acquired MRSA (CA-MRSA) has emerged in recent years (5). CA-MRSA infections, a majority of which in the United States are caused by the highly virulent USA300 genotype (6, 7), often occur in immunocompetent patients lacking obvious risk factors (4). Vancomycin is the drug of choice for treating MRSA infections (8, 9). However, due to its extensive use (5, 9), vancomycin-resistant S. aureus (VRSA) has been increasingly isolated since the first reported vancomycin-intermediate S. aureus (VISA) infection in 1997 (10, 11). The emergence of hypervirulent CA-MRSA and VRSA strains is an alarming threat to public health that highlights the need for an S. aureus vaccine. Others have suggested that prophylactic therapies that target CA-MRSA should include inactivated alpha hemolysin (Hla), since Hla is believed to contribute to the severity of USA300 skin infections (12) and is expressed at high levels by USA300 strains (13). Hla is a secreted toxin that binds to the eukaryotic cell surface protein ADAM10 and then oligomerizes to generate a heptameric pore that is cytolytic to multiple cell types, including erythrocytes and endothelial cells (14). Passive immunization with anti-Hla monoclonal antibodies has been shown to reduce lesion size in a mouse dermonecrosis model (15). Inactivated forms of Hla have been included in vaccine candidates that have been studied in the clinic setting (16). A better understanding of the mechanism(s) of protection for this antigen would improve our ability to evaluate these vaccines. For example, information regarding the immune mechanisms of protection for Hla-based vaccines ensures that appropriate immune responses to vaccination are measured both in preclinical and clinical studies. Moreover, the development of correlates of protection can be useful for monitoring vaccine consistency and the susceptibilities of specific populations postvaccination (17). In this study, we examined the mechanisms of protection of a vaccine based on the genetically inactivated alpha hemolysin mutant HlaH35L, in which histidine 35 was substituted with leucine, in a mouse model of S. aureus skin and soft tissue infections (SSTI). HlaH35L binds to the cell membrane; however, it is unable to form a pore and is therefore not toxic (18, 19). We determined the important role that antibodies play in the protection afforded by active immunization with this genetically inactivated Hla-based vaccine and demonstrate that anti-Hla antibody levels correlate with protection. MATERIALS AND METHODS Ethics statement. For all mouse studies, the protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Center for Biologics Evaluation and Research. Reagents. Purified recombinant HlaH35L was generated as described by Brady et al. (20). Rabbit anti-HlaH35L polyclonal antibody production and IgG purification. The production of Rabbit anti-HlaH35L polyclonal antibody was performed by Cocalico Biologicals, Inc. (Reamstown, PA), subject to their IACUC approval. New Zealand White rabbits (2 to 3 months old, male, 1.8 to 2.3 kg) were immunized with 100 μg HlaH35L at day 0. The rabbits were then immunized again on days 21 and 42 with 50 μg HlaH35L. For each dose, the antigen was diluted to 0.5 ml in 0.9% saline and adsorbed to an equal volume of Alhydrogel (Brenntag Biosector, Frederikssund, Denmark). Bleeds were obtained from the rabbits at days 56 and 70. IgG was purified by affinity chromatography. Rabbit preimmune and immune sera were diluted 1:1 in protein A IgG binding buffer (provided by Thermo Scientific, Rockford, IL) and applied to 5 ml protein A columns (...truncated)