Ocular Immunology and Inflammation
ISSN: 0927-3948 (Print) 1744-5078 (Online) Journal homepage: http://www.tandfonline.com/loi/ioii20
Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis Armando R. Caballero, Davide L. Foletti, Michael A. Bierdeman, Aihua Tang, Angela M. Arana, Adela Hasa-Moreno, Emma Ruth B. Sangalang & Richard J. O’Callaghan To cite this article: Armando R. Caballero, Davide L. Foletti, Michael A. Bierdeman, Aihua Tang, Angela M. Arana, Adela Hasa-Moreno, Emma Ruth B. Sangalang & Richard J. O’Callaghan (2015) Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis, Ocular Immunology and Inflammation, 23:4, 297-303, DOI: 10.3109/09273948.2014.920035 To link to this article: http://dx.doi.org/10.3109/09273948.2014.920035
Published online: 09 Jun 2014.
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Date: 05 November 2015, At: 22:40
Ocular Immunology & Inflammation, 2015; 23(4): 297–303 Copyright ! Taylor & Francis Group, LLC ISSN: 0927-3948 print / 1744-5078 online DOI: 10.3109/09273948.2014.920035
ORIGINAL ARTICLE
Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis
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Armando R. Caballero, PhD1, Davide L. Foletti, PhD2, Michael A. Bierdeman, Aihua Tang, PhD1, Angela M. Arana, BS1, Adela Hasa-Moreno, BS2, Emma Ruth B. Sangalang, BA2, and Richard J. O’Callaghan, PhD1 1
BS
1
,
Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi, USA and 2 Rinat Laboratories, Pfizer Inc., San Francisco, California, USA
ABSTRACT Purpose: To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. Methods: A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. Results: LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p 0.0001). Overall pathology was similar in all groups (p 0.070), but iritis and chemosis were reduced by treatment (p 0.036). Conclusions: The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis. Keywords: Alpha-toxin, erosions, Fab antibody therapy, S. aureus keratitis
Staphylococcus aureus is the primary cause of ocular infections in North America and a leading cause of contact lens-associated keratitis.1–7 S. aureus secretes a variety of toxins that directly attack corneal tissue and indirectly activate the host’s inflammatory response, resulting in the degradation, necrosis, and scarring of the cornea with a concomitant decrease in visual acuity.8–11 Alpha-toxin has been identified as the major virulence factor in keratitis, a finding based on both rabbit and mouse models of infection.8,9,12–14 Alpha-toxin is secreted as a 33-kDa protein, which, after binding to and activating its receptor, ADAM10, can cause either disruption of intercellular junctions or lysis of the cell through the formation of a heptameric structure that creates a pore in the cellular membrane.15–18 Alpha-toxin is known to induce the lysis of a variety of cells, including rabbit
erythrocytes, human platelets, monocytes, and endothelial cells. Alpha-toxin is a potent chemoattractant and a modulator of polymorphonuclear neutrophils, and it can also stimulate the secretion of inflammatory molecules, such as interleukin-1b, interleukin-6, and tumor necrosis factor-a.16,18 Research in this laboratory has shown that the injection of as little as 12 hemolytic units (HU) of alpha-toxin directly into rabbit corneas results in massive pathology characterized by corneal edema, epithelial sloughing, iritis, and conjunctival inflammation.9,19 Furthermore, these studies have shown that S. aureus mutant strains deficient in alpha-toxin cause significantly less pathology in rabbit corneas than their wild-type parental strains or the mutant after complementation of the mutated alpha-toxin gene.20,21
Received 11 December 2013; revised 19 February 2014; accepted 28 April 2014; published online 5 June 2014 Correspondence: Dr. Richard O’Callaghan, 2500 North State St., Jackson, MS 39216, USA. E-mail: [email protected]
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298 A. Caballero et al. Our attempts to develop an effective ocular therapy against this powerful toxin have ranged from immunization with an alpha-toxin toxoid22 to the use of a chemical inhibitor that interferes with the binding of alpha-toxin to its cellular receptor.19,23 These experiments have yielded encouraging results, but improvements in the therapy are needed. The experiments presented herein investigate the possibility of using a high-affinity human monoclonal Fab fragment antibody to alpha-toxin (LTM14 Fab), derived from a human scFv phage library, as topical therapy in a rabbit model of Staphylococcus aureus keratitis. This antibody has been used previously prophylactically and therapeutically in a murine pneumonia, skin abscess, and bacteremia models of infection, in which it proved to be highly effective in providing almost complete protection, a significant decrease in abscess size, and a dramatic increase in survivability of the infected host.24
MATERIALS AND METHODS Chemicals All chemicals and enzymes were obtained from Sigma-Aldrich (St. Louis, MO), unless noted otherwise.
were anesthetized with ketamine hydrochloride and euthanized with an intravenous injection of 1 mL sodium pentobarbital (FatalPlus, Patterson Veterinary Supply, Devers, MA).
Human Monoclonal Fab Fragment Antibody to Alpha-toxin (LTM14 Fab) The alpha-toxin specific monoclonal antibody LTM14 was isolated from a human scFv phage library, and its Fab fragment was generated in HEK293 cells as described by Foletti et al.24
Western Blot Analysis An aliquot (40 mL) of concentrated culture supernatant (10-fold) from a 10-mL overnight culture of S. aureus 8325-4 was electrophoresed in a 12% SDS-PAGE and electroblotted onto nitrocellulose. After incubation in Tris-buffered saline (TBS) with 5% goat serum for 1 h, the membrane was incubated overnight at 4 C with a 1/500 dilution of LTM14 Fab (11.0 mg/mL stock) and developed the next day with a 1/500 dilution of goat anti-human IgG peroxidase conjugate (Capel, Cochranville, PA).
Inhibition of Hemolysis by LTM14 Fab Bacteria Staphylococcus aureus strain 8325-4 was obtained from T. Foster (Department of Microbiology, Trinity College, Dublin, Ireland). This strain has been used extensively in studies of S. aureus keratitis in rabbit and mouse models of infection. S. aureus strain 8325-4 was grown in tryptic soy broth (TSB; Difco, BD Biosciences, Sparks, MD) at 37 C.
The inhibitory effects of various concentrations of LTM14 Fab on the alpha-toxin hemolytic activity were measured by a modification of the procedure previously described by Weeks et al.23 Briefly, 12 HUs of alpha-toxin were incubated with 1:1 serial dilutions of LTM14 Fab (starting concentration of 550 mg/well) and 107 rabbit erythrocytes at 37 C for 45 min. The inhibitory titer of the LTM14 Fab antibody was determined as the highest dilution to prevent at least 50% lysis of erythrocytes.
Animals New Zealand white rabbits 6–8 weeks old were obtained from Charles River Laboratories International (Montreal, Canada). All animals were specific pathogen free and were maintained according to institutional guidelines and tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocols for the animals were approved by the Animal Care Committee of the University of Mississippi Medical Center. Before any procedure was performed, each rabbit was anesthetized by subcutaneously injecting a 1:5 mixture of xylazine (100 mg/mL; Rompum; Miles Laboratories, Shawnee, KS) and ketamine hydrochloride (100 mg/mL; Ketaset; Fort Dodge Animal Health, Fort Dodge, IA). At the end of the experiment, rabbits
Intrastromal Injection of Bacteria Rabbit eyes (10 eyes per group) were topically treated with proparacaine hydrochloride (0.5%, Bausch & Lomb, Tampa, FL) prior to injection. A 10-mL aliquot containing 100 colony-forming units (CFU) of S. aureus strain 8325-4 in TSB was injected intrastromally using a 30-gauge needle as previously described.25
LTM14 Fab Antibody Therapy Rabbit eyes were treated with a single topical drop (45 mL, 11.0 mg/mL) of LTM14 Fab antibody to Ocular Immunology & Inflammation
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incubated at 37 C for 24 h. CFUs per cornea were determined and expressed as base 10 logarithms.
alpha-toxin alone, or LTM14 Fab mixed with 0.006% benzalkonium chloride (BAK, Sigma Aldrich), every 30 min beginning at 9 h post-infection (PI) until 14 h PI (11 drops total, 495 mg of LTM14 Fab/drop). As a negative control, infected eyes were also treated with a nonspecific Fab fragment antibody. The nonspecific Fab was obtained from a monoclonal IgG specific for bovine herpes virus.
Statistical Analysis Statistical analysis of SLE scores and erosion area was performed with Kruskal-Wallis one-way analysis of variance. CFU determinations were analyzed with Student’s t test. p 0.05 was considered significant.
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Slit-lamp Examination RESULTS
Rabbit eyes underwent slit-lamp examination (SLE) at 15 h PI by two masked observers using a Topcon biomicroscope, and photographs were taken. Each of seven parameters (injection, chemosis, iritis, hypopyon, corneal infiltrate, fibrin in the anterior chamber, corneal edema) was graded on a scale ranging from 0 (normal) to 4 (severe). The sum of these grades for an eye, after averaging, determined the SLE score, which could range from 0 to a theoretical maximum of 28. Erosion size was measured in mm2 under a blue filter after staining with fluorescein.
Western blot analysis of the concentrated culture supernatant from an overnight culture of S. aureus strain 8325-4 with LTM14 Fab antibody to alpha-toxin identified a 33-kDa protein band identical to the protein band seen in the adjacent lane containing purified alpha-toxin (Figure 1A, lanes 1 and 2, respectively). A hemolysis inhibition assay demonstrated that LTM14 Fab had an inhibition titer of 4096 against 12 HU of alpha-toxin (Figure 1B). These data confirm the specificity of the antibody as well as its toxin neutralizing activity. The effectiveness of LTM14 Fab antibody therapy in the treatment of S. aureus 8325-4 keratitis was next examined. Two different treatment strategies were used: LTM14 Fab alone and LTM14 Fab + BAK. BAK is a common preservative in ophthalmic preparations that has been shown to increase ocular permeability to drugs by disruption of tight junctions between corneal epithelial cells.26,27
Quantification of Viable Bacteria Rabbits were sacrificed at 15 h PI and their corneas were harvested and homogenized in 3.0 mL sterile phosphate-buffered saline (PBS). A 0.1-mL aliquot of the homogenate was serially diluted 1:10 in PBS and plated on tryptic soy agar in triplicate. Plates were (A)
KDa
1
2
37
25
(B)
FIGURE 1. Western blot analysis and hemolysis inhibition assay using a high-affinity human Fab fragment of antibody to alpha-toxin. (A) Western blot analysis of concentrated culture supernatant from S. aureus strain 8325-4 (lane 1) and purified alpha-toxin (lane 2) Copyright ! 2015 Taylor & Francis Group, LLC
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FIGURE 2. Effect on erosion area of Fab antibody to alpha-toxin with or without BAK. (A) Erosion area (mm2) present in untreated eyes (˙), eyes treated with LTM14 Fab antibody to alpha-toxin (g), and eyes treated with LTM14 Fab + BAK (m) at 15 h PI (n 14). (B) Photographs of erosions after staining with fluorescein at 15 hours PI. *Statistically significant from untreated control; zstatistically significant from each other.
As shown in Figure 2, antibody therapy starting at a time point when signs of infection were clearly visible (9 h PI) was very effective in limiting the size of the corneal epithelial erosions, which are a direct effect of the alpha-toxin secreted by S. aureus. The untreated controls had erosions averaging 30.67 mm2 (±1.833), the eyes treated with LTM14 Fab had erosions averaging 16.92 mm2 (±1.807), and the eyes treated with LTM14 Fab plus BAK had erosions
averaging 11.01 mm2 (±0.959). There was a statistical difference between untreated and treated eyes (p 0.001), and between treatment with antibody alone or antibody plus BAK (p = 0.036). The antibody therapy did not interfere in any way with the growth of S. aureus strain 8325-4 in the rabbit cornea (Figure 3). Untreated eyes contained 6.871 log10 CFU (±0.136) by the end of the experiment, whereas eyes treated with LTM14 Fab had Ocular Immunology & Inflammation
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a-Toxin Fab Antibody Therapy for S. aureus Keratitis
FIGURE 3. Bacterial load (in log10 CFU) present in infected corneas. Eyes were untreated (˙), treated with LTM14 Fab antibody to alpha-toxin (g), or treated with LTM14 Fab + BAK (m) at 15 h PI (n 10).
6.880 (±0.188) log10 CFU, and eyes treated with LTM14 Fab plus BAK had 6.690 (±0.111) log10 CFU. There was no statistical difference between the groups (p 0.114). This was not surprising, since the antibody was not expected to interfere with bacterial growth. Treatment of infected eyes with a Fab fragment antibody against bovine herpes virus with or without BAK resulted in erosion areas of 31.17 (±3.447) and 31.09 (±2.159) mm2, respectively, and log10 CFUs of 6.792 (±0.154) and 6.856 (±0.122), respectively, that were not significantly different from the untreated controls (p 0.310). Although the antibody therapy significantly limited the formation of toxin-mediated epithelial erosions, the therapy did not significantly reduce the overall SLE scores (Figure 4A). SLE scores were 9.75 (±0.369) for the untreated controls, 8.69 (±0.552) for the eyes treated with LTM14 Fab, and 8.52 (±0.499) for the eyes treated with LTM14 Fab plus BAK (p 0.070). However, both the overall appearance (Figure 4B) and a statistical analysis indicate that the eyes treated with the antibody exhibited less pathology in some of the parameters that contribute to the overall SLE score. The chemosis score for the untreated eyes was 1.95 (±0.109) vs. 1.43 (±0.145) for the eyes treated with LTM14 Fab plus BAK (p = 0.002), and for iritis, the untreated eyes had a parameter score of 3.49 (±0.083) vs. 3.22 (±0.090) for the eyes treated with LTM14 Fab alone (p = 0.036) and 2.99 (±0.092) for the eyes treated with LTM14 Fab plus BAK (p = 0.001).
CONCLUSION Staphylococcus aureus is one of the most important human pathogens due to its ability to cause infection in a variety of tissues and body sites. Of particular Copyright ! 2015 Taylor & Francis Group, LLC
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importance are ocular infections which, if misdiagnosed or not treated promptly, can lead to loss of visual acuity or blindness. The major virulence factor in S. aureus keratitis is the hemolysin alphatoxin.8,9,12–14 This protein is responsible not only for de-anchoring the epithelial layer of the cornea from the stroma, but also for being a potent chemoattractant and a trigger of inflammatory cytokines.9,16 Previous experiments have shown that systemic immunization with an alpha-toxin toxoid did have a beneficial effect in terms of lessening ocular pathology when rabbit eyes were infected with S. aureus.22 This finding suggested that a high-affinity antibody against alpha-toxin could be used topically to treat S. aureus keratitis. However, one early concern was that a full-sized antibody molecule could have limited penetration into the corneal stroma, and thus would lack effectiveness. For this reason, the Fab portion of the human monoclonal antibody LTM14, a very high-affinity (1.7pM) neutralizing antibody specific for alpha-toxin,24 was tested alone or in combination with BAK, an agent that increases corneal permeability.26,27 The therapy with LTM14 Fab alone or with BAK was effective in significantly limiting the size of the corneal erosions as compared to the untreated control or eyes treated with a nonspecific Fab with or without BAK. Furthermore, the eyes treated with antibody plus BAK had significantly smaller erosions than the eyes treated with antibody alone, indicating that BAK allowed for better penetration of the Fab antibody fragment into the corneal tissue. The possibility exists that BAK may have had a direct effect on ocular pathology, perhaps by influencing bacterial viability or the activity of alpha-toxin. We consider this unlikely because, at the concentrations used, BAK had no effect in the CFUs recovered from the homogenized corneas as shown in Figure 3. Furthermore, BAK with a nonspecific Fab antibody had no effect on the size of the erosions or the SLE scores. BAK’s mode of action is at the corneal surface and not in the stroma, where the bacteria are located. In addition, in vitro hemolysis inhibition assays using the Fab antibody in the presence or absence of BAK showed no difference in the hemolysis inhibition titer (data not shown). The overall ocular pathology as measured by the numerical SLE scores did not show a statistically significant difference between the treated and untreated groups. This can be explained by the contribution of several factors. The LTM14 Fab treatment was specific for alpha-toxin, thus it had no effect on the bacterial population that was constantly renewing the supply of active alphatoxin. The effects of the toxin on the corneal epithelium were effectively inhibited by the antibody Fab fragment, but those effects occurring at more internal sites were less exposed to the topically
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FIGURE 4. Effect on SLE scores of Fab antibody to alpha-toxin with or without BAK. (A) SLE scores of untreated eyes (˙), eyes treated with LTM14 Fab antibody to alpha-toxin (g), and eyes treated with LTM14 Fab + BAK (m) at 15 h PI (n 14). (B) Photographs of ocular pathology at 15 h PI.
applied therapy. Cells distant from the corneal surface, yet exposed to the toxin being secreted by the bacteria in the corneal stroma, could be active in secreting cytokines and other mediators that affect other ocular sites. It is important to remember that the overall SLE score incorporates analysis of the conjunctiva, cornea, and anterior chamber. One must also take into account the fact that, although alpha-toxin is a major virulence factor in the eye, S. aureus also secretes beta- and gamma-toxins, various cysteine and serine proteases, superantigens,
and superantigen-like proteins, all of which could contribute to the overall pathology of the infection. Although there were no statistical differences in the SLE scores between treated and untreated groups, there was a significant difference in some of the parameters that contribute toward the overall score, i.e. iritis and chemosis. These could be due to the direct effect of the antibody neutralizing alpha-toxin near the immediate surface of the eye. Chemosis, which is the accumulation of extracellular fluid in conjunctival tissue, and iritis, which is caused by the Ocular Immunology & Inflammation
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a-Toxin Fab Antibody Therapy for S. aureus Keratitis
distention of the iris and the release of inflammatory cells from iris capillaries, are well-known side effects of purified alpha-toxin injection into the cornea.9 The antibody treatment clearly interfered, at this particular time point, with the degree of pathology exhibited at these locations. The rest of the parameters seem to be unaffected and this could be due to the inability of the antibody to reach the toxin present at deeper sites, or to the action of additional virulence factors, or the concomitant immune response to the growing infection. In summary, the high-affinity human LTM14 Fab fragment antibody against alpha-toxin, either alone or formulated with BAK, was able to interfere with the ability of alpha-toxin to cause corneal erosions. This therapy benefits the infected eye and the possibility exists that a combination therapy of Fab with antibiotic would have a dramatic effect on the overall ocular pathology.
DECLARATION OF INTEREST Davide Foletti, Adela Hasa-Moreno, and Emma Sangalang were employees and shareholders of Pfizer Inc. at the time the study was conducted.
REFERENCES 1. Govindarajan B, Menon BB, Spurr-Michaud S, et al. A metalloproteinase secreted by Streptococcus pneumoniae removes membrane mucin MUC16 from the epithelial glycocalyx barrier. PLoS One. 2012;7:e32418. 2. Liesegang TJ. Bacterial keratitis. In Kaufman HE, Baron BA, McDonald MB (eds.), The Cornea, 2nd ed. Boston: Butterworth-Heineman; 1998:159–218. 3. Wong VW, Lai TY, Chi SC, Lam DS. Pediatric ocular surface infections: a 5-year review of demographics, clinical features, risk factors, microbiological results, and treatment. Cornea. 2011;30:995–1002. 4. Jeng BH, Gritz DC, Kumar AB, et al. Epidemiology of ulcerative keratitis in Northern California. Arch Ophthalmol. 2010;128:1022–1028. 5. Kerr N, Stern GA. Bacterial keratitis associated with vernal keratoconjunctivitis. Cornea. 1992;11:355–359. 6. Palmer ML, Hyndiuk RA. Contact lens-related infectious keratitis. Int Ophthalmol Clin. 1993;33:23–49. 7. Sowka JW, Gurwood AS, Kabat AG. Handbook of Ocular Disease Management Web Site: Bacterial Keratitis. New York, NY: Jobson; 2001. 8. Callegan MC, Engel LS, Hill JM, O’Callaghan RJ. Corneal virulence of Staphylococcus aureus: roles of a-toxin and protein A in pathogenesis. Infect Immun. 1994;62:2478–2482. 9. O’Callaghan RJ, Callegan MC, Moreau JM, et al. Specific roles of a-toxin and b-toxin during Staphylococcus aureus corneal infection. Infect Immun. 1997;65:1571–1578.
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10. Projan S, Novick R. The molecular basis of pathogenicity. In Crossley K, Archer G (eds.), The Staphylococci in Human Disease. New York: Churchill Livingston; 1999:55–82. 11. Chusid MJ, Davis SD. Experimental bacterial keratitis in neutropenic guinea pigs: polymorphonuclear leukocytes in corneal host defense. Infect Immun. 1979;24:948–952. 12. Girgis DO, Sloop GD, Reed JM, O’Callaghan RJ. Effects of toxin production in a murine model of Staphylococcus aureus keratitis. Invest Ophthalmol Vis Sci. 2005;46: 2064–2070. 13. O’Callaghan RJ, Callegan MC, Moreau JM, et al. Specific roles of a-toxin and b-toxin during Staphylococcus aureus corneal infection. Infect Immun. 1997;65:1571–1578. 14. Moreau JM, Sloop GD, Engel LS, et al. Histopathological studies of staphylococcal a-toxin: effects on rabbit corneas. Curr Eye Res. 1997;16:1221–1228. 15. Dinges MM, Orwin PM, Schlievert PM. Exotoxins of Staphylococcus aureus. Clin Microb Rev. 2000;13:16–34. 16. Bownik A, Siwicki AK. Effects of staphylococcal hemolysins on the immune system. Centrl Eur J Immunol. 2008;33: 87–90. 17. Powers ME, Kim HK, Wang Y, Bubeck WJ. ADAM10 mediates vascular injury induced by Staphylococcus aureus alpha hemolysin. J Infect Dis. 2012;206:352–356. 18. Berube BJ, Wardenburg JB. Staphylococcus aureus a-toxin: nearly a century of intrigue. Toxins. 2013;5: 1140–1166. 19. McCormick CC, Caballero AR, Balzli CL, et al. Chemical inhibition of alpha-toxin, a key corneal virulence factor of Staphylococcus aureus. Invest Ophthalmol Vis Sci. 2009;50: 2848–2854. 20. Dajcs JJ, Austin MS, Sloop GD, et al. Corneal pathogenesis of Staphylococcus aureus strain Newman. Invest Ophthalmol Vis Sci. 2002;43:1109–1115. 21. Dajcs JJ, Thibodeaux BA, Girgis DO, O’Callaghan, RJ. Corneal virulence of Staphylococcus aureus in an experimental model of keratitis. DNA Cell Biol. 2002;21: 375–382. 22. Hume EB, Dajcs JJ, Moreau JM, O’Callaghan RJ. Immunization with alpha-toxin toxoid protects the cornea against tissue damage during experimental Staphylococcus aureus keratitis. Infect Immun. 2000;68: 6052–6055. 23. Weeks AC, Balzli CL, Caballero AR, et al. Identification and potency of cyclodextrin-lipid inhibitors of Staphylococcus aureus a-toxin. Curr Eye Res. 2012;37:87–93. 24. Foletti D, Strop P, Shaughnessy L, et al. Mechanism of action and in vivo efficacy of a human-derived antibody against Staphylococcus aureus a-hemolysin. J Mol Biol. 2013; 425:1641–1654. 25. Hobden JA, Reidy JJ, O’Callaghan RJ, et al. Treatment of experimental Pseudomonas keratitis using collagen shields containing tobramycin. Arch Ophthalmol. 1988;106: 1605–1607. 26. Wensheng C, Zhiyuan L, Jiaoyue H, et al. Corneal alternations induced by topical application of benzalkonium chloride in rabbit. PLOS One. 2011;6:1–8. 27. Romanowski EG, Mah FS, Kowalski RP, et al. Benzalkonium chloride enhances the antibacterial efficacy of gatifloxacin in an experimental rabbit model of intrastromal keratitis. J Ocular Pharm Therapeutics. 2008; 24:380–384.
ISSN: 0927-3948 (Print) 1744-5078 (Online) Journal homepage: http://www.tandfonline.com/loi/ioii20
Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis Armando R. Caballero, Davide L. Foletti, Michael A. Bierdeman, Aihua Tang, Angela M. Arana, Adela Hasa-Moreno, Emma Ruth B. Sangalang & Richard J. O’Callaghan To cite this article: Armando R. Caballero, Davide L. Foletti, Michael A. Bierdeman, Aihua Tang, Angela M. Arana, Adela Hasa-Moreno, Emma Ruth B. Sangalang & Richard J. O’Callaghan (2015) Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis, Ocular Immunology and Inflammation, 23:4, 297-303, DOI: 10.3109/09273948.2014.920035 To link to this article: http://dx.doi.org/10.3109/09273948.2014.920035
Published online: 09 Jun 2014.
Submit your article to this journal
Article views: 70
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ioii20 Download by: [York University Libraries]
Date: 05 November 2015, At: 22:40
Ocular Immunology & Inflammation, 2015; 23(4): 297–303 Copyright ! Taylor & Francis Group, LLC ISSN: 0927-3948 print / 1744-5078 online DOI: 10.3109/09273948.2014.920035
ORIGINAL ARTICLE
Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis
Downloaded by [York University Libraries] at 22:40 05 November 2015
Armando R. Caballero, PhD1, Davide L. Foletti, PhD2, Michael A. Bierdeman, Aihua Tang, PhD1, Angela M. Arana, BS1, Adela Hasa-Moreno, BS2, Emma Ruth B. Sangalang, BA2, and Richard J. O’Callaghan, PhD1 1
BS
1
,
Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi, USA and 2 Rinat Laboratories, Pfizer Inc., San Francisco, California, USA
ABSTRACT Purpose: To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. Methods: A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. Results: LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p 0.0001). Overall pathology was similar in all groups (p 0.070), but iritis and chemosis were reduced by treatment (p 0.036). Conclusions: The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis. Keywords: Alpha-toxin, erosions, Fab antibody therapy, S. aureus keratitis
Staphylococcus aureus is the primary cause of ocular infections in North America and a leading cause of contact lens-associated keratitis.1–7 S. aureus secretes a variety of toxins that directly attack corneal tissue and indirectly activate the host’s inflammatory response, resulting in the degradation, necrosis, and scarring of the cornea with a concomitant decrease in visual acuity.8–11 Alpha-toxin has been identified as the major virulence factor in keratitis, a finding based on both rabbit and mouse models of infection.8,9,12–14 Alpha-toxin is secreted as a 33-kDa protein, which, after binding to and activating its receptor, ADAM10, can cause either disruption of intercellular junctions or lysis of the cell through the formation of a heptameric structure that creates a pore in the cellular membrane.15–18 Alpha-toxin is known to induce the lysis of a variety of cells, including rabbit
erythrocytes, human platelets, monocytes, and endothelial cells. Alpha-toxin is a potent chemoattractant and a modulator of polymorphonuclear neutrophils, and it can also stimulate the secretion of inflammatory molecules, such as interleukin-1b, interleukin-6, and tumor necrosis factor-a.16,18 Research in this laboratory has shown that the injection of as little as 12 hemolytic units (HU) of alpha-toxin directly into rabbit corneas results in massive pathology characterized by corneal edema, epithelial sloughing, iritis, and conjunctival inflammation.9,19 Furthermore, these studies have shown that S. aureus mutant strains deficient in alpha-toxin cause significantly less pathology in rabbit corneas than their wild-type parental strains or the mutant after complementation of the mutated alpha-toxin gene.20,21
Received 11 December 2013; revised 19 February 2014; accepted 28 April 2014; published online 5 June 2014 Correspondence: Dr. Richard O’Callaghan, 2500 North State St., Jackson, MS 39216, USA. E-mail: [email protected]
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298 A. Caballero et al. Our attempts to develop an effective ocular therapy against this powerful toxin have ranged from immunization with an alpha-toxin toxoid22 to the use of a chemical inhibitor that interferes with the binding of alpha-toxin to its cellular receptor.19,23 These experiments have yielded encouraging results, but improvements in the therapy are needed. The experiments presented herein investigate the possibility of using a high-affinity human monoclonal Fab fragment antibody to alpha-toxin (LTM14 Fab), derived from a human scFv phage library, as topical therapy in a rabbit model of Staphylococcus aureus keratitis. This antibody has been used previously prophylactically and therapeutically in a murine pneumonia, skin abscess, and bacteremia models of infection, in which it proved to be highly effective in providing almost complete protection, a significant decrease in abscess size, and a dramatic increase in survivability of the infected host.24
MATERIALS AND METHODS Chemicals All chemicals and enzymes were obtained from Sigma-Aldrich (St. Louis, MO), unless noted otherwise.
were anesthetized with ketamine hydrochloride and euthanized with an intravenous injection of 1 mL sodium pentobarbital (FatalPlus, Patterson Veterinary Supply, Devers, MA).
Human Monoclonal Fab Fragment Antibody to Alpha-toxin (LTM14 Fab) The alpha-toxin specific monoclonal antibody LTM14 was isolated from a human scFv phage library, and its Fab fragment was generated in HEK293 cells as described by Foletti et al.24
Western Blot Analysis An aliquot (40 mL) of concentrated culture supernatant (10-fold) from a 10-mL overnight culture of S. aureus 8325-4 was electrophoresed in a 12% SDS-PAGE and electroblotted onto nitrocellulose. After incubation in Tris-buffered saline (TBS) with 5% goat serum for 1 h, the membrane was incubated overnight at 4 C with a 1/500 dilution of LTM14 Fab (11.0 mg/mL stock) and developed the next day with a 1/500 dilution of goat anti-human IgG peroxidase conjugate (Capel, Cochranville, PA).
Inhibition of Hemolysis by LTM14 Fab Bacteria Staphylococcus aureus strain 8325-4 was obtained from T. Foster (Department of Microbiology, Trinity College, Dublin, Ireland). This strain has been used extensively in studies of S. aureus keratitis in rabbit and mouse models of infection. S. aureus strain 8325-4 was grown in tryptic soy broth (TSB; Difco, BD Biosciences, Sparks, MD) at 37 C.
The inhibitory effects of various concentrations of LTM14 Fab on the alpha-toxin hemolytic activity were measured by a modification of the procedure previously described by Weeks et al.23 Briefly, 12 HUs of alpha-toxin were incubated with 1:1 serial dilutions of LTM14 Fab (starting concentration of 550 mg/well) and 107 rabbit erythrocytes at 37 C for 45 min. The inhibitory titer of the LTM14 Fab antibody was determined as the highest dilution to prevent at least 50% lysis of erythrocytes.
Animals New Zealand white rabbits 6–8 weeks old were obtained from Charles River Laboratories International (Montreal, Canada). All animals were specific pathogen free and were maintained according to institutional guidelines and tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocols for the animals were approved by the Animal Care Committee of the University of Mississippi Medical Center. Before any procedure was performed, each rabbit was anesthetized by subcutaneously injecting a 1:5 mixture of xylazine (100 mg/mL; Rompum; Miles Laboratories, Shawnee, KS) and ketamine hydrochloride (100 mg/mL; Ketaset; Fort Dodge Animal Health, Fort Dodge, IA). At the end of the experiment, rabbits
Intrastromal Injection of Bacteria Rabbit eyes (10 eyes per group) were topically treated with proparacaine hydrochloride (0.5%, Bausch & Lomb, Tampa, FL) prior to injection. A 10-mL aliquot containing 100 colony-forming units (CFU) of S. aureus strain 8325-4 in TSB was injected intrastromally using a 30-gauge needle as previously described.25
LTM14 Fab Antibody Therapy Rabbit eyes were treated with a single topical drop (45 mL, 11.0 mg/mL) of LTM14 Fab antibody to Ocular Immunology & Inflammation
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incubated at 37 C for 24 h. CFUs per cornea were determined and expressed as base 10 logarithms.
alpha-toxin alone, or LTM14 Fab mixed with 0.006% benzalkonium chloride (BAK, Sigma Aldrich), every 30 min beginning at 9 h post-infection (PI) until 14 h PI (11 drops total, 495 mg of LTM14 Fab/drop). As a negative control, infected eyes were also treated with a nonspecific Fab fragment antibody. The nonspecific Fab was obtained from a monoclonal IgG specific for bovine herpes virus.
Statistical Analysis Statistical analysis of SLE scores and erosion area was performed with Kruskal-Wallis one-way analysis of variance. CFU determinations were analyzed with Student’s t test. p 0.05 was considered significant.
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Slit-lamp Examination RESULTS
Rabbit eyes underwent slit-lamp examination (SLE) at 15 h PI by two masked observers using a Topcon biomicroscope, and photographs were taken. Each of seven parameters (injection, chemosis, iritis, hypopyon, corneal infiltrate, fibrin in the anterior chamber, corneal edema) was graded on a scale ranging from 0 (normal) to 4 (severe). The sum of these grades for an eye, after averaging, determined the SLE score, which could range from 0 to a theoretical maximum of 28. Erosion size was measured in mm2 under a blue filter after staining with fluorescein.
Western blot analysis of the concentrated culture supernatant from an overnight culture of S. aureus strain 8325-4 with LTM14 Fab antibody to alpha-toxin identified a 33-kDa protein band identical to the protein band seen in the adjacent lane containing purified alpha-toxin (Figure 1A, lanes 1 and 2, respectively). A hemolysis inhibition assay demonstrated that LTM14 Fab had an inhibition titer of 4096 against 12 HU of alpha-toxin (Figure 1B). These data confirm the specificity of the antibody as well as its toxin neutralizing activity. The effectiveness of LTM14 Fab antibody therapy in the treatment of S. aureus 8325-4 keratitis was next examined. Two different treatment strategies were used: LTM14 Fab alone and LTM14 Fab + BAK. BAK is a common preservative in ophthalmic preparations that has been shown to increase ocular permeability to drugs by disruption of tight junctions between corneal epithelial cells.26,27
Quantification of Viable Bacteria Rabbits were sacrificed at 15 h PI and their corneas were harvested and homogenized in 3.0 mL sterile phosphate-buffered saline (PBS). A 0.1-mL aliquot of the homogenate was serially diluted 1:10 in PBS and plated on tryptic soy agar in triplicate. Plates were (A)
KDa
1
2
37
25
(B)
FIGURE 1. Western blot analysis and hemolysis inhibition assay using a high-affinity human Fab fragment of antibody to alpha-toxin. (A) Western blot analysis of concentrated culture supernatant from S. aureus strain 8325-4 (lane 1) and purified alpha-toxin (lane 2) Copyright ! 2015 Taylor & Francis Group, LLC
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FIGURE 2. Effect on erosion area of Fab antibody to alpha-toxin with or without BAK. (A) Erosion area (mm2) present in untreated eyes (˙), eyes treated with LTM14 Fab antibody to alpha-toxin (g), and eyes treated with LTM14 Fab + BAK (m) at 15 h PI (n 14). (B) Photographs of erosions after staining with fluorescein at 15 hours PI. *Statistically significant from untreated control; zstatistically significant from each other.
As shown in Figure 2, antibody therapy starting at a time point when signs of infection were clearly visible (9 h PI) was very effective in limiting the size of the corneal epithelial erosions, which are a direct effect of the alpha-toxin secreted by S. aureus. The untreated controls had erosions averaging 30.67 mm2 (±1.833), the eyes treated with LTM14 Fab had erosions averaging 16.92 mm2 (±1.807), and the eyes treated with LTM14 Fab plus BAK had erosions
averaging 11.01 mm2 (±0.959). There was a statistical difference between untreated and treated eyes (p 0.001), and between treatment with antibody alone or antibody plus BAK (p = 0.036). The antibody therapy did not interfere in any way with the growth of S. aureus strain 8325-4 in the rabbit cornea (Figure 3). Untreated eyes contained 6.871 log10 CFU (±0.136) by the end of the experiment, whereas eyes treated with LTM14 Fab had Ocular Immunology & Inflammation
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a-Toxin Fab Antibody Therapy for S. aureus Keratitis
FIGURE 3. Bacterial load (in log10 CFU) present in infected corneas. Eyes were untreated (˙), treated with LTM14 Fab antibody to alpha-toxin (g), or treated with LTM14 Fab + BAK (m) at 15 h PI (n 10).
6.880 (±0.188) log10 CFU, and eyes treated with LTM14 Fab plus BAK had 6.690 (±0.111) log10 CFU. There was no statistical difference between the groups (p 0.114). This was not surprising, since the antibody was not expected to interfere with bacterial growth. Treatment of infected eyes with a Fab fragment antibody against bovine herpes virus with or without BAK resulted in erosion areas of 31.17 (±3.447) and 31.09 (±2.159) mm2, respectively, and log10 CFUs of 6.792 (±0.154) and 6.856 (±0.122), respectively, that were not significantly different from the untreated controls (p 0.310). Although the antibody therapy significantly limited the formation of toxin-mediated epithelial erosions, the therapy did not significantly reduce the overall SLE scores (Figure 4A). SLE scores were 9.75 (±0.369) for the untreated controls, 8.69 (±0.552) for the eyes treated with LTM14 Fab, and 8.52 (±0.499) for the eyes treated with LTM14 Fab plus BAK (p 0.070). However, both the overall appearance (Figure 4B) and a statistical analysis indicate that the eyes treated with the antibody exhibited less pathology in some of the parameters that contribute to the overall SLE score. The chemosis score for the untreated eyes was 1.95 (±0.109) vs. 1.43 (±0.145) for the eyes treated with LTM14 Fab plus BAK (p = 0.002), and for iritis, the untreated eyes had a parameter score of 3.49 (±0.083) vs. 3.22 (±0.090) for the eyes treated with LTM14 Fab alone (p = 0.036) and 2.99 (±0.092) for the eyes treated with LTM14 Fab plus BAK (p = 0.001).
CONCLUSION Staphylococcus aureus is one of the most important human pathogens due to its ability to cause infection in a variety of tissues and body sites. Of particular Copyright ! 2015 Taylor & Francis Group, LLC
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importance are ocular infections which, if misdiagnosed or not treated promptly, can lead to loss of visual acuity or blindness. The major virulence factor in S. aureus keratitis is the hemolysin alphatoxin.8,9,12–14 This protein is responsible not only for de-anchoring the epithelial layer of the cornea from the stroma, but also for being a potent chemoattractant and a trigger of inflammatory cytokines.9,16 Previous experiments have shown that systemic immunization with an alpha-toxin toxoid did have a beneficial effect in terms of lessening ocular pathology when rabbit eyes were infected with S. aureus.22 This finding suggested that a high-affinity antibody against alpha-toxin could be used topically to treat S. aureus keratitis. However, one early concern was that a full-sized antibody molecule could have limited penetration into the corneal stroma, and thus would lack effectiveness. For this reason, the Fab portion of the human monoclonal antibody LTM14, a very high-affinity (1.7pM) neutralizing antibody specific for alpha-toxin,24 was tested alone or in combination with BAK, an agent that increases corneal permeability.26,27 The therapy with LTM14 Fab alone or with BAK was effective in significantly limiting the size of the corneal erosions as compared to the untreated control or eyes treated with a nonspecific Fab with or without BAK. Furthermore, the eyes treated with antibody plus BAK had significantly smaller erosions than the eyes treated with antibody alone, indicating that BAK allowed for better penetration of the Fab antibody fragment into the corneal tissue. The possibility exists that BAK may have had a direct effect on ocular pathology, perhaps by influencing bacterial viability or the activity of alpha-toxin. We consider this unlikely because, at the concentrations used, BAK had no effect in the CFUs recovered from the homogenized corneas as shown in Figure 3. Furthermore, BAK with a nonspecific Fab antibody had no effect on the size of the erosions or the SLE scores. BAK’s mode of action is at the corneal surface and not in the stroma, where the bacteria are located. In addition, in vitro hemolysis inhibition assays using the Fab antibody in the presence or absence of BAK showed no difference in the hemolysis inhibition titer (data not shown). The overall ocular pathology as measured by the numerical SLE scores did not show a statistically significant difference between the treated and untreated groups. This can be explained by the contribution of several factors. The LTM14 Fab treatment was specific for alpha-toxin, thus it had no effect on the bacterial population that was constantly renewing the supply of active alphatoxin. The effects of the toxin on the corneal epithelium were effectively inhibited by the antibody Fab fragment, but those effects occurring at more internal sites were less exposed to the topically
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FIGURE 4. Effect on SLE scores of Fab antibody to alpha-toxin with or without BAK. (A) SLE scores of untreated eyes (˙), eyes treated with LTM14 Fab antibody to alpha-toxin (g), and eyes treated with LTM14 Fab + BAK (m) at 15 h PI (n 14). (B) Photographs of ocular pathology at 15 h PI.
applied therapy. Cells distant from the corneal surface, yet exposed to the toxin being secreted by the bacteria in the corneal stroma, could be active in secreting cytokines and other mediators that affect other ocular sites. It is important to remember that the overall SLE score incorporates analysis of the conjunctiva, cornea, and anterior chamber. One must also take into account the fact that, although alpha-toxin is a major virulence factor in the eye, S. aureus also secretes beta- and gamma-toxins, various cysteine and serine proteases, superantigens,
and superantigen-like proteins, all of which could contribute to the overall pathology of the infection. Although there were no statistical differences in the SLE scores between treated and untreated groups, there was a significant difference in some of the parameters that contribute toward the overall score, i.e. iritis and chemosis. These could be due to the direct effect of the antibody neutralizing alpha-toxin near the immediate surface of the eye. Chemosis, which is the accumulation of extracellular fluid in conjunctival tissue, and iritis, which is caused by the Ocular Immunology & Inflammation
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distention of the iris and the release of inflammatory cells from iris capillaries, are well-known side effects of purified alpha-toxin injection into the cornea.9 The antibody treatment clearly interfered, at this particular time point, with the degree of pathology exhibited at these locations. The rest of the parameters seem to be unaffected and this could be due to the inability of the antibody to reach the toxin present at deeper sites, or to the action of additional virulence factors, or the concomitant immune response to the growing infection. In summary, the high-affinity human LTM14 Fab fragment antibody against alpha-toxin, either alone or formulated with BAK, was able to interfere with the ability of alpha-toxin to cause corneal erosions. This therapy benefits the infected eye and the possibility exists that a combination therapy of Fab with antibiotic would have a dramatic effect on the overall ocular pathology.
DECLARATION OF INTEREST Davide Foletti, Adela Hasa-Moreno, and Emma Sangalang were employees and shareholders of Pfizer Inc. at the time the study was conducted.
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