yduosnuew Jouny YerHIN yduosnue w Joumny YerHIN yduosnue W JouINY Wd-HIN Mo, @: eo NIH Public Access Author Manuscript Published in final edited form as: Expert Opin Ther Targets, 2012 June ; 16(6): 601-612. doi:10.1511N14728222.2012.682573, The potential use of toxin antibodies as a strategy for controlling acute Staphylococcus aureus infections Gordon Y. C. Cheung, Ph.D. and Laboratory of Human Bacterial Pathogenesis, NIAID, NIH, Bldg. 33, Room 1W10, 9000 RocWille Pike, Bethesda, MD 20892, USA, Tel. +1 301 443 5211 Michael Otto, Ph.D. Laboratory of Human Bacterial Pathogenesis, NIAID, NIH, Bldg. 33, Room 1W10, 9000 Rocwille Pike, Bethesda, MD 20892, USA, Tel. +1 301 443 5209 Gordan ¥.C. Cheung: cheunggo@niaid nih gov; Michael Otto: motto@niaid.nih.gov Abstract Introduction—The pandemic human pathogen, Staphylococcus aureus displays high levels of antibiotic resistence and is @ major cause of hospital. and community-associated infections. S ‘aweusdisease manifestation is fo a great extent due to the production ofa large axsenal of virulence factors, which include a seties of secreted toxins. Antibodies to S. auweus toxins are founcl in people who are infected or asymptometically colonized with S. aureus Immunotherepies consisting of neutralizing anti-toxin antibodies could provide immediate eid to patients with paired immune systems or in advanced stages of disease Areas covered—Important S: eureustoxins, their roles in pathogenesis, rationales for selecting ‘S. aureus toxins for immunization efforts, and caveats associated with monoclonal antibody-based passive immunization are discussed, This review will focus on hyper-virulent community- associated methicillin resistant S- aurcus(CA-MRSA) because oftheir ecent surge and clinical importance. Expert opinion—Antbodies ageinst genome-encoded toxins maybe more broadly applicable than those directed against toxins found only ina sub-population of. aureusisolates Furthermore, there is substantial functional redundancy among S: aureustoxins. Thus, an optimal anti-S. aweusfommulation may consist of multiple antibodies directed ageinst a series of hey 5. ‘aweusgenome-encoded toxins. Keywords. Toxin, vaccine; immunotherapy, methicillin-resistant Staphylococcus aureus, monoclonal antibody, alpha-hemolysin, phenol-soluble modulins, superantigens, leukotoxins; Panton- Valentine leukocidin 4. The need for immunotherapies and vaccines against Staphylococcus aureus ‘The commensal microorganism, Staphylococcus aureus is a primary colonizer of human ares (in ~ 25% of the population) and the etiological agent of many diseases 1). Since the Corespandence to: Michael Oto,mottoCniaid nih. gov. ‘Declaration of Interest: “his wank was sopperted bythe rural search Program ofthe Nationa tne of Alergy and Infectious Diseases, NLyduosnuew Joumny YeHIN yduosnue W Jonny Va-HIN qduosnuew Jouany Wd-HIN Cheng O89 Page? 1960s, the use of antibiotics to treat S: aureusassociated infections has become increasingly problematic owing to the emergence of methicillin-resistant S' aureus(MRSA) and S ‘aurcusstains that are resistant to a wide variety of different antibiotics 3). Originally athogen primarily of immune-compromised and otherwise predisposed patents in hospital settings (hospital associated- MRSA; HA-MRSA) (1, more aggressive methicillin-resistant strains ofS aueushave recently appeared in the community (community-essociated- MRSA, CA-MRSA), which may successfully infect healthy human people U4. The ajonty of CA-MRSA infections are soft skin and tissue infections (SSTIs) (~ 50% of total cases), However, invasive cases of CA-MRSA-related diseases, such as necrotizing ‘meumonia (~ 5% of total cases), are becoming increasingly more widespread. As @ consequence, the treatment of MRSA-infections places great financial stains on public healthcare [1 Itis commonly believed that the epidemiological success of the pulsed-field type US A300 CA-MRSA isolate, which is now pandemic in the United States, is due to @ combination of high virulence, antibiotic resistance, and colonization capacity leading to sustainable spread. in the community (1, The high mumber of community-essociated infections with USA300 ‘as prompted considerable research efforts aimed to develop therapeutics to combat CA- (MRSA. These primarily include strategies interfering with virulence, including most notably active and passive immunization efforts directed against CA-MRSA toxins and other ‘varulence factors (reviewed extensively in 19), Here, an overview one nunber of $\ aureustoxin families, witha focus on S: eureustoxins encoded exclusively by, or showing increased expression in, CA-MIRSA strains will be ‘provided. The roles that those toxins have in S. aureuspathogenesis and disease will be discussed. Finally the rationales and caveats regarcing the use of enti-toxin monoclonal ‘antibodies (mAbs) as therapeutics for the prevention and treatment of CA-MRSA-mediated disease will be explored. 2. Toxins involved in S. aureus pathogenesis S aureusproduces and secretes many types of toxins with diverse roles in pathogenesis, ‘auticulerly affecting immune evasion and activation ofthe immune response. These include alpha-hemolysin (a-hemolysin, Hla), beta-toxin (f-toxin), the supsrantigens (SAgs), the Jeukotoxins, and the phenol-soluble modulins (PSIMs). The genes coding for f-toxin, the ‘SAgs and leukotoxins are mostly encoded by mobile genetic elements (MGEs) 01), therefore, toxin expression can differ tremendously between different stains of S. eweus Onlya few 5: eureustoxins, such as a-hemolysin and the PSMs, are genome- encoded 2,13] and expressed byboth HA- and CA-MRSA strains. However, a-hemolysin and the PSMs are expressed more strongly in CA-MRS A compared to HA-MRSA strains. ‘This suggests that differences in gene regulation may influence the epidemiological success of CA-MRSA strains such as USA300 151, For example, it has been noted that USA30O shows increased expression of the global regulatory quorum sensing system, accessory gene regulator (ag? 19), which controls the expression of many virulence factors in a cell density- cependent manner. The agrlocus consists of a divergent promoter, which controls the transcription of RNAII and RNAIII, The RNAII transcript contains 4 genes encoding a classical two-component quorum sensing system (agrAC), an auto-inducing peptide (AIP) recttsor (agrD), and the AIP maturation end exporter protein (ag). Upon increasing concentration of AIP during growth, the AgrAC two-component system becomes activated, ata certain threshold concentration of AIP. Upon binding of AIP to the membrane protein AgiC, AgrA is activated, which in tum binds to the divergent promoter and induces transcription of RNAI and RNAIII. RNAI isa regulatory RNA molecule that regulates the expression of e majonty of toxins Ul: aweusmutans lacking a functional age regulatory Espert Opin Ter Tange, Acthar mamscrpt eveiablen PMC 2013 Kae 02yduosnuew Joumny YeHIN yduosnue W Jonny Va-HIN qduosnuew Jouany Wd-HIN Page system are significantly less virulent than the comesponding wild-type strains, which has recently been demonstrated specifically for USA300 [17-19 2.4 Leukotoxins ‘Neutrophils, or polymorphonuclear leukocytes (PINs), are key components of the innate immune system and involved in controlling 5° aureusinfection 9,20) Possibly for that reason, 5’ eureusproduces a large variety of virulence factors that inhibit neutrophil function 2, allowing S: aweusto circumvent elimination by innate host defense 221 Particularly, pore-forming, bi-component leukotoxins with cytolytic affinity towards cells of ‘myeloid lineage, such as monocytes, macrophages and neutrophils, represent key contributors to S$ aureusimmune evasion 25) Rach leukotoxin requires one class § and one class F protein, which are individually non-toxic, to form a G-barreled pore-forming structure upon oligomerization [1 Six class S subunits (LukS-PV, Higa, HigC, LukE, LukM, LukH) and five class F subunits (LukF-PV, HigB, LukD, LukF’-PV, LukG) have been described (#,25.27.25], One exception to this monogamous pring is the y-hemolysin gene cluster, which comprises three genes ((higA [or hig’, hig [or fuk} and higB [or TukAl), whose gene products allow the formation of two functional pairs of proteins: Hig [High + HigB] end Luk (HigB + HigC]. Notably, all leukotoxins reportedly contribute to disease progression in atleast some animal infection models, withthe exception of LukM Lak” py Boat While the Afggene cluster occurs in99% of S: eureusstrains ©, many other leukotoxin genes are not uniformly present among 5: aueusisolates. The /ukDE O°) and JuGH (ukAB) °.25) genes canbe found in many sequenced strains or clinica isolates including USA300 4, However, for example, fukDE are not found in the HA-MRSA isolate, USA200 P61 andl the phage-encoded JukF-PY and lulS-PY 03), which code for Panton- Valentine leukocidin (PVL), are mostly restricted to CA-MRSA strains 07), represented by only ~ 5% of ll clinical isolates. Ofnote, the role of PVL in S- eureuspathogenesis remains controversial despite extensive research that has been performed on that Jeukotoxin (15,3838 Owing to the initially observed epidemiological link of sukS'F-P” with CA-MRSA, it has been suggested that PVL contributes to the pathogenesis of typical CA.MRSA disease manifestations, such as necrotizing pmewmonia [540 or the formation of shin abscesses 2,42 However, PVL-negetive CA-IMRSA strains have since been isolated, indicating that this epidemaiological conelation isnot absolute 2.15), Furthermore, ‘animal infection studies investigating the role of PVL in S. aweusdisease axe not in ‘agreement 4-50 These discrepancies may in pautbe due to differences ofthe animal ‘models used, such as inoculum size andl host species. Over the last decade, the PVL. controversy may have overshaclowed the importance of other, and pethaps more relevant, Jeukotoxins tht are expressed by a majorityof CA-MRSA stains, such as JukDE end JukGH. These leukotoxins axe now being investigated more intensely. Studies using isogenic S aureusdeletion mutants show that LukDE and LukGH contribute tothe virulence of 5: aureusin murine sepsis 241 ancl renal abscess models 2, respectively. Furthermore, there have been some, albeit relatively few studies on the contribution of -hemolysin to 5: aureus pathogenesis and disease. These studies indicate that y-hemolysin may have roles in septic arthritis and weight loss in mice [9 and endophthalmitis in rabbits 1, However, itis ‘unlaown whether LukDE, LukGH or y-hemolysin contribute towards other facets of HA- and CA-MRSA disease, for example, severe cases of pmeumonia, as has been suggested for PUL. 22. Staphylococcal superantigens ‘The superantigen (S.Ag) family consists of small, serologically distinct proteins including staphylococcal enterotoxins (SEs) A-E, G-l, staphylococcal enterotoxin-like toxins (SEI) Espert Opin Ter Tange, Acthar mamscrpt eveiablen PMC 2013 Kae 02yduosnuew Joumny YeHIN yduosnue W Jonny Va-HIN qduosnuew Jouany Wd-HIN Cheng O89 Paget XR, U end X, and Toxic Shock Syndrome Toxin-1 (TSST-1), all of which are ~ 20-28 Da in size (¥.53,54 Members of the SAg family aze responsible for food poisoning, toxic shock syncrome and respiratory disease (°1. The SAgs share a common protein structuze [3.4 that enables each SAg to crosslink different alleles of major ‘histocompatibility complex (MHC) class II molecules on the surfaces of antigen-presenting cells with diferent alleles of the variable region of T-cell eceptors (TCRs) (7. These interactions allow the SAgs to bypass the conventional antigen processing pathway to non- specifically activate T-cells (~ 50%) and induce the imegular production of high levels of| inflammatory cytokines (60) This event induces multisystem disease that includes rash, ‘hypotension, pyrexia, emesis and diarrhea (611 The SAgs also cause severe life-threatening smltiple organ failze (1) (Of the lange mumber of SAgs that have been described, only the recently discovered SELX is genome-encoded [) all other SAgs are encoded by MGEs '©2-S41, Moreover, not all S ‘aurcusisolates display superantigenic activity 5 because the SAgs are not distabuted equallyamong S' eucusstrains 65, Furthermore, its not unusual to see varied levels of ‘SAg production among S aureusstrains that express SAgs (67-72) This is attributed to the involvement of at least three global regulators, agr 3), sazA 4, o? and saeRS 5. Interestingly, TSST-1 and SEB have been reported to act as negative global regulators of, toxin production 1 ‘The roles of SAgs in the pathogenesis of 5: aurcusdsease have been investigated in animal ‘models by aduinistration of purified SAgs (71. Other studies have compared the differences in the capacities of natwally occunning SAg* and SAg” strains ("] and isogenic SAg mutants [5,792 to cause disease in animals. The overall importance of the SAgs in CA- MRSA pathogenesis is debatable, because many Ags that are classicelly associated with S- aureusdisease are not expressed by CA-MIRSA strins. For instance, USA300 does not typically produce TSST-1, SEB or SEC, SAgs that are most often associated with toxic shock (3.221, As one noticeable exception, SEIX has been shown to contribute to USA300 necrotizing pneumonia in rabbits [, However, its unknown if SELK contributes to other manifestations of CA-MRSA disease, such as SSTIs. 23. Alpha-hemolysin “The genome-encoded a-hemolysin (Hla), one of the first toxins described for S. aureus has recently received considerable attention asa target for an anti-S: aureusvaccine and the ‘production of neutralizing antibodies for passive immunization. q-hemolysin is produced by ‘a majonityof S. aweusstrains and its expression is regulated by at least thee global regulatory systems, which include eg ©), tis well known for its pore-forming (3,°#-29] ‘and pro-inflammatory 91 properties. However, its cognate receptor, the zinc-dependent retaloprotease ADAMO (A disintegrin and metalloproteinase 10), was only recently iscovered ®1,921_ q hemolysin contnbutes significantly tothe prthogenesis of S. aureus USA300-induced skin infection and pneumonia '6.°3.°4, Mice that do not express ADAMIO are resistant to lethal infection with S- aureusstrain USA300 in a pneumonia ‘model, but tis not known if ADAMO is also requized for a-hemolysin induced abscess formationby 5: aureus Another signal, which is found on imumune cells, may also be required for a-hemolysin-induced pneumonia by S- eurcus 5], This second signal belongs to a class of cytosolic proteins called nucleotide-binding domain and leucine-rich repeat containing (NLR) proteins, which are important activators ofthe innate immune response NLRs defend ageinst pathogen infection and encogenous damage in response to a variety of| ‘mimunogenic stimuli 1, Upon activation, several members ofthis family assemble into lange multimeric protein complexes celled inflammasomes, which induce inflammatory responses. NLRP3 in patcular is activated in response to bacterial toxins ©), including 5. aureusf-toxin, y-hemolysin ©] and q-hemolysin ©), Thus, NLPR3 inflammasome Espert Opin Ter Tange, Acthar mamscrpt eveiablen PMC 2013 Kae 02yduosnuew Joumny YeHIN yduosnue W Jonny Va-HIN qduosnuew Jouany Wd-HIN Pages activation and the presence of ADAMO appear to contribute to the development of S. aureus pneumonia 2.4 Phenol-soluble modulins ‘The phenol-soluble modulins (PSMs) are a family of small, amphipathic peptides with cytolytic and pro-inflammatory properties towards @ range of cells types including erythrocytes and neutrophils (2. The pro-inflammatory properties of PSMs (such as, induction of intracellular calcium flux, chemotaxis, and cytokine production in neutrophils) ‘are dependent on binding to their cognate receptor, human formyl peptide receptor 2 (FPR2). Of note, PSM-dependent cel lysis occurs independently of FPR2 binding ©, Two PSMsuib-families canbe distinguished: PSMa peptides, which include the well-known &- toxin, are 20-26 amino acids, and PSIMp peptides 43 ~ 44 amino acids in length 221 Generally, members of the PSMa class show more pronounced! pro-inflammatory ancl especially lytic activities than members of the PSM class [21 ‘Similar to many other S- aureusvirulence determinants, PSMs are under control of the ag global regulatory system (2.200), However, in contrast to most Agr targets, they are regulated dzectly by AgrA rather than vie RNAAIII 200,10]. Most S: eweusstains contain the RNAlI[-embedded Afdgene encoding 6-toxin 11, and the operons coding for the psa ‘and the psnfigenes, which are located on two separate loci [3. In contrast to the ubiquitous prevalence of the psma and the panfigenes, the psm-mec gene 93] is restricted to HA- [MRSA strains carrying staphylococcal cessette chromosome mec(SCCzed) types Il, Ill and vin ou) Importantly, CA-MRSA isolates secrete higher amounts of PSMBs in average compared to HA.MRSA isolates (21, suggesting that the PSM have @ prominent role in CA-MRSA_ pathogenesis. Indeed, isogenic CA-MRSA pana mutants ae severely impaired in their bility to form abscesses and cause septs in mice (2, Collectively, the a-cless PSMs, including 6-toxin, and the @-class PSM all play azole in the dissemination of CA-MRSA ‘fom biofilm-related catheter infections in mace 4, Biofilms are sticky agglomerations consisting of a mixture of bacterial cells and extracellular polysaccharide matrices end axe intrinsically esstant to antibiotics ane mechanisms of host defense [5], The detergent-ike quality of PSMs (2.206 influences the development of mature biofilms by breaking interactions between cell populations, inoducing channel formation, followed by the detachment of cell clusters, leading to bacterial dissemination in vivo 004,107]. Finally, PSMs act synergistically with ptoxin [98,1091 and PVL (91 in vito. Since important CA- ‘MRSA isolates do not produce -toxin (1) and not all CA-MRSA isolates hasbor the /ut- PV and JutS-PY genes, this synexgism may infer a cooperative relationship between, -vrulence factors produced by different strains of 5: eureusthat have different toxin expression profiles. 3. Anti-toxin antibody responses in humans Soon after birth, 40 - 50% of the population cany S: aureus By 14 months of age, there can be a~ 40% reduction in cariage rates (12.1131 and S? aureuscan remain a persistent colonizer in the nares of adults in ~ 25% of the population (41. In healthy human individuals, antibody responses toward many S' aurcusantigens can be detected, such as cell surface components, non-protein antigens, and toxins (5.113118), indicating that S' aureus antigens are immunogenic Antibodies against most 5: aureustoxins are not readily detected in healthy newboms although ant-TSST-1 antbodies were reported [1. In healthy adults, camiers of S. aureus can produce greater levels of antibodies towards 5: aureusantigens than non-cartiers, but Espert Opin Ter Tange, Acthar mamscrpt eveiablen PMC 2013 Kae 02yduosnuew Joumny YeHIN yduosnue W Jonny Va-HIN qduosnuew Jouany Wd-HIN Cheng O89 Pages these titers are generally less pronounced at the age of 65 and higher (161. Of the anti-toxin antibodies, only significantly higher levels of ant-TSST-1 and anti-SEA have been described in cazziers compared to non-carziers, and itis thought that previous exposure(s) to different S. sureusstrains may account for the diverse levels of antibody titers between individuals [16,07 Individuals infected with S: aureuegenerally produce greeter levels of antibodies to PVL, a- hemolysin, and SAgs than healthy individuals (4125, 419,120) Furthermore, low levels of ‘antibodies maybe associated with more severe S. aweusingections (9), However, presence of antibodies is not necessarily associated with protection, as recently described forthe role of anti-PVL antibodies in PVL+CA-MRSA infections in iumans (21 Sem fiom 5 aweusinfected patents may demonstrate neutralizing activity ageinst S aweur toxing, sch es SAgs (27,122,125 hut whether there is neutrlizing eetivityof these sera towards other 5. aueustoxins remains tobe determined. Altogether, although much information hes been garnered from these serological studies, the roles that anti-S. aureus antodies play during infection stil remains lngely unknown, 4. S. aureus toxins as targets for monoclonal antibody therapy Unlike active immunization, which sometimes requires repeated boosters and long pesiods of time for maximum immune responses to be generated, passive immunization would provide immediate treatment for unvaccinated patients to help reduce the severity of acute S saureurdisease. Pethaps even more importantly, mAb therepy would benefit mmune- compromised patients or infants with immature immune systems or even ada tothe existing ool of potentially protective anti-S: eureurantibodies. Cleazly itis important for such therapy to take effect that S. aweuris rapidly identified. Moreover, late stages of S. aureus infection may not be amenable to passive therapy, as advanced stages of toxin-mediated tissue destruction will have ensued. Even though there are caveats to passive immunization, much research has recently been performed on neutralizing mAbs against S. aweustoxins, ‘and some of these successfully protected fiom S: aueusinféction in animal models (Table 1), Polyclonal anti-toxin antibodies also are protective in animal models of S- aureus infection (Table 1). However, many ofthe investigated toxin, such as PVL and SEB, are not expressed by all. aweusstains. Therefore, anti-PVL and anti-SEB antibodies may onlybe useful in treating acute infections caused by PVL and SEB-expressing S. aweus strains. Instead, core-genome encoded toxins, sch as a-hemolysin, SELX, LukGH, and PSMs maybetter serve as targets for antibody generation, as antibodies ageinst those toxins maybe more broadly applicable for acute cases of S: aureurinfections. The use of these toxins as targets for mAb generation willbe discussed in more detail below. 4.1 Antibody therapy against the leukotoxins Even though there is controversy over whether PVL is a decisive virulence determinant in S aureus there is stil song interest in creating a vaccine against PVL end producing ent PVL mAbs, There ate several points supporting the use of ent-PVL antibodies for passive ‘immunization. Fist, humans are capable of generating humoral immune responses against PVL [115,117,121] Second, polyclonal and mAbs to PVL are neutralizing {21,12¢,125] ‘Third, injection of PVL together with a tetravalent ant-PVL mAb, which binds both LukS- PV end LukF-PV antigens simultaneously, reduced inflammation and tissue destruction in @ non-lethal model of endophthalmitis in rbbits 124). Finally, ant-PVL mAbs exhibit cross- reactivity with other leukotoxins, such as HigC 041. On the other hand, there are many ‘important findings that negate the benefits of using anti-PVL antibodies. Fist, polyclonal anti-PVL antibodies failed to neutralize the cytotoxicity of a PVL-expressing USA300 strain toward human neutrophils invite (5, Second, anti-PVL immune sera did not protect from Espert Opin Ter Tange, Acthar mamscrpt eveiablen PMC 2013 Kae 02yduosnuew Joumny YeHIN yduosnue W Jonny Va-HIN qduosnuew Jouany Wd-HIN Cheng O89 Pag ‘S aureus peumonia in passive immunization experiments using mice 1, Third, PVL- neutalizing antibodies did not protect children from primary or recuzent CA-MRSA- associated SSTis (20, Finally, the presence of anti-PVL anbbotes may in fact exacerbate disease caused by PVL-expressing CA-MRSA during early stages of infection 0271, These ‘antibodies may prevent the interactions of PVL with cells ofthe innate immune system, resulting in poor activation of the immune response and a decreased ability to mount an attack to the infection LukDE and LukGH (LukAB) also interact with innate immune cells 2.25) and are lytic toward neutrophils 24.251 Furthermore, PVL and LukGH lyse other innate immune cells, such as human monocytes and macrophages 25.1281. qureusstrains that express one or ‘ore of these leukotoxins at sufficient levels thus may severely impr the bility ofthe host to mount an effective immune response. However, itemains to be investigated whether ‘antibodies to leukotoxins other than PVL also mayenhance the virulence of S: aureusin a way simular to ant-PVL antibodies. Finally, it has been reported that active immunization. with PVL subunits relieves mice of severe skin and intranasal infection with PVL- expressing CA-MRSA M4, Phase II clinical trials axe curently underway using @ recombinant LukS-PV toxin subunit, The results ofthis study have yet o be released (1291 ‘Taken together, there are strong arguments against the use of anti-PVL antibodies to treat CALMRSA disease. Furthermore, there is cuently no compelling evidence for a necessary role of PVL in CA-MRSA pathogenesis, leaving the possiblity that CA-MRSA strains without PVL may further expand or PVL-positive CA-MRSA strains may lose the PVL- ‘encoding prophage without a substantial loss of virulence characteristics. Moreover, ‘pursuing development of an anti-PVL mAb- or antigen-based vaccine is problematic from @ marketing point of view, because only~ 5% of 5 aureusclinical isolates possess the genes coding for PVL, limiting its applicability. Finally, although active immunization with PVL subunits appears to be an encouraging alternative to passive immunization, active ‘vaccination with a combination of LukGH or LukDE subunits may help protect against a lnger number of S* eureusinfections 4.2 Antibody therapy against the superantigens ‘The SAgs are responsible fora range of symptoms and diseases in humans that range from food poisoning to toxic shock. Passive therapy with ant-S.Ag antibodies would likely help alleviate the symptoms associated with SAg-related disease by preventing the interactions of the SAgs with their ligands. Antibodies against TSST-1, SEA, SEB, SEC are readily cetected in healthy individuals (17,150-132 and individuals with active S. aureus infections 1), indicating that these SAgs are immunogenic. ‘There is particular interest in developing neutralizing antibodies against the category B bioterrorism egent, SEB, which is resistant to denaturing and highly toxic to ‘humans 133,154], Ant-SEB mAbs are neutralizing in vitro 1351 and passive therapy with ‘anti SEB antbodies successfully prevents SEB-induced disease manifestations in mice [36] ‘and monkeys (171 against lethal challenge with purified SEB. While there is certainly cross- reactivity of anti-SAg mAbs with several SAgs (131, tis unlikely that a monovalent SEB ‘mAb will protect from infection by many SEB" expressing 5: aureusstrains of high clinical ‘importance, such as the pandemic CA-MRSA strains. In contrast, passive immunuzation with several different anti-SAg mAbs would potentially be able to protect against a larger variety of S. eweusstrains. Furthermore, the genome-encoded SEIX may serve as an attractive altemative target for generating mAbs, because it is expressed by a majority of S. aurcusisolates including the CA-MRSA strain USA300 (4, Finally, passive immunization 1s not the only strategy for interfering with SAg-producing strains; other strategies include active immunization with detoxified SAgs (3°14 the use of soluble forms of engineered Espert Opin Ter Tange, Acthar mamscrpt eveiablen PMC 2013 Kae 02yduosnuew Joumny YeHIN yduosnue W Jonny Va-HIN qduosnuew Jouany Wd-HIN Cheng O89 Pages TCR V6 proteins 1), and hybrid TCR Vp/MHCI proteins 11 that can block the binding activity of SEB toward its natural ligands found on T cells and antigen-presenting cells. 4.3 Antibody therapy against «hemolysin Healthy human individuals that are colonized with S. aureus produce high levels of ‘antibodies directed toward a-hemolysin 16,1171. As q-hemolysin is expressed by a ajonty of 5: aureusstrains, passive immunization with mAbs to a-hemolysin maybe effective against many different S' eureusstrains including strains of CA-MRSA. Several studies have shown the protective efficacy of anti-a-hemolysin antibodies in S. aureus abscess [4,145] and pneumonia 6,146) murine infection models. In addition, active ‘immunization witha detoxified a-hemolysin derivative (H35L) was shown to reduce the severity of S aureusinduced pneumonia (41 and 5. aureusabscess (51 infections Interestingly, recombinant a-hemolysin toxoid is being tested in the same phase II clinical tral as recombinant LukS-PV 0291 4.4 Antibody therapy against the phenol-soluble modulins ‘The genome-encoded PSMs show litle variation in amino acid sequences between S' aureus strains (1, Therefore, antibodies against PSMs could serve as an effective passive ‘immunization strategy against a broad spectrum of S: eureusstrains. Infants with invasive 5. aureusinfections do not appear to produce antibodies to the strongly cytolytic PSMa3 {0471 but anti-PSMentibodies canbe raised in animals injected with PSMs and adjuvant [197,148] indicating that the PSMs have immunogenic potental. High levels of PSMs are produced by CA-MRSA strains in vito 12.141, but the levels of PSMin-vivo production are yet ‘unknown, Therefore, the lack of enti-PSM antibody production, which is cbserved atleast in infants, may either be due to a low production level of PSIMs under those conditions, or if the infection is localized, such as ina SSTI, the host may not be eble to detect enough PSMs ‘in order to generate antibodies. Antibodies against PSIMs maybe more readily detected in adults or in other types of St aureusdiseases. However, this needs to be determined by including the relatively recently discovered PSMSs as antigens in future human serological studies Although S. aureuscatheter-related biofilm infections are not a focus of this review, this subject area warrants a brief discussion. PSMs playa crucial role in biofilm structuring and lissemination of biofilm-associated infection in these organisms 004.107]. Notably, anti- PSMp antibodies blocked the dissemination of S. epidermictecatheter infection in mice ‘Therefore, passive immunization with anti-PSM antibodies maybe useful to inhibit the dissemination of bacteria from biofilm formed on catheters. However, more research is clearly needed to investigate antibody therapy against PSM, 5. Expert opinion ‘There is an wgent need for research aimed to find a successful immunization strategy against S. auwreusinfections. This is warranted in particular because ofthe high levels of antibiotic resistance shown by pandemic strains of aureus. Antibodies against S. aureus toxins may have particular value, as they neutralize the binding affinity of the toxins, preventing ro-inflammatory responses and cytolytic activities. Furthermore, they may block toxin oligomerization even after insertion into cell membranes, thus preventing cytolytic activity of the toxin Choosing a specific S. eureustoxin as a singular vaccine taxget is complicated by the fact that 5. aureusproduces many, often functionally redundant foxins. Some toxins are immunogenic and play important roles in S. aureuspathogenesis, but many are not expressed by all S. aureusisolates. Therefore, targeting core-genome encoded toxins, such Espert Opin Ter Tange, Acthar mamscrpt eveiablen PMC 2013 Kae 02yduosnuew Joumny YeHIN yduosnue W Jonny Va-HIN qduosnuew Jouany Wd-HIN Cheng O89 Pages as a-hemolysin, LulGH, SEIX or the PSMs, would serve asa better basis for vaccine development than toxins only produced by a sub-population of stains, suchas PVL,, ‘TSST-1, SEA, SEB and SEC, especially when aiming fora vaccine prepaetion with broad applicability ‘Monoclonal antibodies raised against only one 5: aurcusantigen are protective in animal models of S. aureusinfection, but fequently, only disappointing results were achieved with zmanyof those mAbs in human phase 1/2 clinical tials reviewed in 4). An alternative, ‘and perhaps more successful, strategy would comprise a mAb preparation consisting of ‘antibodies against different toxins and possibly other S. aweurvirulence factors, sich as surface proteins, to simultaneously target establishment and exacerbation of infection. In theory, such antibody therapy would thus be able to inhibit diferent aspects of S. aweus pathogenesis. Interestingly, it was recently shown that administration of two different mAbs ‘against aureusSEB led to significantly more protection than one singular SEB.cirected mAb (135, This concept s not new - simular results have been achieved with mAbs against toxins produced by other bacterial species, such as Bacillus enthracisanthrax toxin 1, Clostridium botulinum newotoxin A (1 and C: cificife toxins A and B !21. However, it needs tobe further explored whether such a vaccination strategy, ie. using antibodies aginst different S. aweurantigens, would be broadly effective against S. aweus Moreover, itneeds to be cautiously evaluated whether antitoxin antibodies may have the opposite effect of exacerbating 5. aureusdisease, suchas in the case of passive immunization with ant- PVL antibodies (27) Insummary, unsuccessfl clinica tials with predominantly non-toxin-based S* aureus antigens warrant new approaches to protect against the new wave of pandemic CA-MRSA strains in addition to the continuously high disease burden due to HA-MRSA. There is strong evidence from laboratory experiments suggesting that passive immunization approaches using anti-toxin antibodies maybe es successful as those directed toward other S aureusantigens. 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