Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens.

Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A ß-(1→6)-linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule is synthesized by four proteins encoded in genetic loci designated intercellular adhesion in Staphylococcus aureus or polyglucosamine in selected Gram-negative bacterial pathogens. We report that many microbial pathogens lacking an identifiable intercellular adhesion or polyglucosamine locus produce PNAG, including Gram-positive, Gram-negative, and fungal pathogens, as well as protozoa, e.g., Trichomonas vaginalis, Plasmodium berghei, and sporozoites and blood-stage forms of Plasmodium falciparum. Natural antibody to PNAG is common in humans and animals and binds primarily to the highly acetylated glycoform of PNAG but is not protective against infection due to lack of deposition of complement opsonins. Polyclonal animal antibody raised to deacetylated glycoforms of PNAG and a fully human IgG1 monoclonal antibody that both bind to native and deacetylated glycoforms of PNAG mediated complement-dependent opsonic or bactericidal killing and protected mice against local and/or systemic infections by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, Candida albicans, and P. berghei ANKA, and against colonic pathology in a model of infectious colitis. PNAG is also a capsular polysaccharide for Neisseria gonorrhoeae and nontypable Hemophilus influenzae, and protects cells from environmental stress. Vaccination targeting PNAG could contribute to immunity against serious and diverse prokaryotic and eukaryotic pathogens, and the conserved production of PNAG suggests that it is a critical factor in microbial biology.

Microbiota-driven immune cellular maturation is essential for antibody-mediated adaptive immunity to Staphylococcus aureus infection in the eye.

As an immune-privileged site, the eye, and particularly the outer corneal surface, lacks resident mature immune effector cells. Physical barriers and innate mediators are the best-described effectors of immunity in the cornea. When the barriers are breached, infection can result in rapid tissue destruction, leading to loss of visual acuity and frank blindness. To determine the cellular and molecular components needed for effective adaptive immunity on the corneal surface, we investigated which immune system effectors were required for protection against Staphylococcus aureus corneal infections in mice, which are a serious cause of human eye infections. Both systemically injected and topically applied antibodies to the conserved cell surface polysaccharide poly-N-acetylglucosamine (PNAG) were effective at mediating reductions in corneal pathology and bacterial levels. Additional host factors impacting protection included intercellular adhesion molecule 1 (ICAM-1)-dependent polymorphonuclear leukocyte (PMN) recruitment, functional CD4(+) T cells, signaling via the interleukin-17 (IL-17) receptor, and IL-22 production. In germfree mice, there was no protective efficacy of antibody to PNAG due to the lack of LY6G(+) inflammatory cell coeffector recruitment to the cornea. Protection was manifest after 3 weeks of exposure to conventional mice and acquisition of a resident microbiota. We conclude that in the anterior eye, ICAM-1-mediated PMN recruitment to the infected cornea along with endogenous microbiota-matured CD4(+) T cells producing both IL-17 and IL-22 is required for antibody to PNAG to protect against S. aureus infection.

Topical neutralization of interleukin-17 during experimental Pseudomonas aeruginosa corneal infection promotes bacterial clearance and reduces pathology.

The proinflammatory cytokine interleukin-17 (IL-17) is involved in neutrophilic tissue infiltration, contributing to both microbial clearance as well as inflammation-associated tissue damage. Its role during bacterial corneal infections is unknown. We hypothesized that IL-17 responses would be detrimental in this setting and tested the impact of IL-17 receptor deficiency or antibody-mediated neutralization of IL-17 in a murine model of Pseudomonas aeruginosa ulcerative keratitis after scratch injury. We found that, compared with infected corneas from wild-type mice, those from IL-17 receptor (IL-17R)-deficient mice had significantly lower corneal pathology scores, neutrophil influx, and intracellular bacterial levels. Infected IL-17R-deficient corneas had low intercellular adhesion molecule 1 (ICAM-1) expression, and ICAM-1-deficient mice were similarly resistant to infection. Topical treatment with polyclonal antibodies to IL-17 resulted in significant reductions in corneal pathology and also lowered bacterial counts after infection with six different laboratory or clinical P. aeruginosa strains, including both invasive and cytotoxic strains. Thus, neutralization of IL-17 during P. aeruginosa corneal infection reduces neutrophil influx and pathology without compromising bacterial clearance and offers a promising new avenue for therapy of these potentially sight-threatening infections.

Disruption of CFTR-dependent lipid rafts reduces bacterial levels and corneal disease in a murine model of Pseudomonas aeruginosa keratitis.

PURPOSE: Pseudomonas aeruginosa enters corneal epithelial cells in vitro via membrane microdomains or lipid rafts. Bacterial entry, mediated by the cystic fibrosis transmembrane conductance regulator (CFTR), promotes infection and disease. This study was conducted to determine whether P. aeruginosa and CFTR are colocalized to rafts in isogenic corneal cells expressing wild-type (WT) or mutant DeltaF508-CFTR and whether disruption of the rafts both in vitro and in vivo affects the bacterial levels and the course of the disease. METHODS: Transformed human corneal epithelial cells from a patient homozygous for DeltaF508-CFTR, and the same cells corrected with WT-CFTR, were exposed to six isolates of P. aeruginosa-three invasive and three cytotoxic strains-in the presence of beta-cyclodextrin (CD), which disrupts rafts. Association and cellular uptake of the invasive strains were measured, as was lactate dehydrogenase release induced by the cytotoxic strains. Scratch-injured mouse eyes were infected with the six P. aeruginosa strains, and the effect of prophylactic or therapeutic administration of CD on bacterial levels and disease was evaluated. RESULTS: P. aeruginosa and CFTR were colocalized with lipid rafts in cells with WT-CFTR, and CD treatment of these cells disrupted bacterial association, internalization, and cytotoxic effects. Cells expressing DeltaF508-CFTR were marginally affected by CD. Prophylactic and therapeutic topical application of CD ameliorated corneal disease and reduced the bacterial count in the eye. CONCLUSIONS: P. aeruginosa enters human corneal epithelial cells via lipid rafts containing CFTR, and disruption of raft-mediated uptake of this organism by CD protects against disease and reduces bacterial levels in the mouse model of keratitis.

Antibodies to Conserved Surface Polysaccharides Protect Mice Against Bacterial Conjunctivitis.

Purpose: Bacterial conjunctivitis is a major problem in ocular health. Little is known about protective immune effectors in the conjunctiva. We evaluated whether opsonic antibody to the conserved surface/capsular polysaccharide poly-N-acetyl glucosamine (PNAG) expressed by Streptococcus pneumoniae and Staphylococcus aureus was protective against bacterial conjunctivitis, as well as an antibody to the Pseudomonas aeruginosa surface polysaccharide alginate. Methods: Bacteria were injected directly into the conjunctivae of either A/J mice or into conjunctivae of wild type C57Bl/6 mice for comparisons to responses of recombination activating gene 1-knock out (RAG 1 KO) or germ-free mice in the C57Bl/6 genetic background. Human IgG1 monoclonal antibodies (MAb) to either PNAG or alginate were administered as follows: direct injection of 10 µg into the conjunctivae or topical application onto the cornea 4, 24, and 32 hours post infection; or intraperitoneal injection of 200 µg 18 hours prior to and then 4, 24, and 32-hours postinfection. After 48 hours, eyes were scored for pathology, mice were euthanized, and CFU/conjunctiva was determined. Results: All methods of antibody administration reduced S. pneumoniae, S. aureus, or P. aeruginosa pathology and bacterial levels in the conjunctivae. Histopathologic analysis showed severe inflammatory cell infiltrates in conjunctivae of mice treated with control MAb, whereas immune mice showed only very mild cellular infiltration. The protective effect of MAb to PNAG was abolished in RAG 1 KO and germ-free mice. Conclusions: Antibodies to both PNAG and alginate demonstrated therapeutic efficacy in models of S. pneumoniae, S. aureus, and P. aeruginosa conjunctivitis, validating the protective capacity of antibodies to surface polysaccharides in distinct ocular tissues.

Efficacy of Antibody to PNAG Against Keratitis Caused by Fungal Pathogens.

Purpose: Developing immunotherapies for fungal eye infections is a high priority. We analyzed fungal pathogens for expression of the surface polysaccharide, poly-N-acetyl glucosamine (PNAG), and used a mouse model of ocular keratitis caused by Aspergillus flavus, A. fumigatus, or Fusarium solani to determine if PNAG was an immunotherapy target and requirements for ancillary cellular and molecular immune effectors. Methods: Enzyme-linked immunosorbent assay (ELISA) or immunofluorescence was used to detect PNAG on fungal cells. Keratitis was induced by scratching corneas of C57BL/6, IL-17R KO, RAG-1 KO, or IL-22 KO mice followed by inoculation with fungal pathogens. Goat antibodies to PNAG, a PNAG-specific human IgG1 monoclonal antibody, or control antibodies were injected either prophylactically plus therapeutically or therapeutically only, and corneal pathology and fungal levels determined in infected eyes at 24 or 48 hours after infection. Results: All tested fungal species produced PNAG. Prophylactic or therapeutic treatment by intraperitoneal (IP) injection of antibody to PNAG combined with post-infection topical application of antibody, the latter also used for A. fumigatus, led to reduced fungal levels, corneal pathology, and cytokine expression. Topical administration only of the PNAG monoclonal antibodies (MAb) reduced fungal loads and corneal pathology. There was no antibody protection in IL-17R KO, RAG-1 KO, or IL-22 KO mice. Conclusions: Poly-N-acetyl glucosamine is produced by clinically important fungal ocular pathogens. Antibody to PNAG demonstrated protection against Aspergillus and Fusarium keratitis, requiring T cells producing IL-17 and IL-22. These findings indicate the potential to prevent or treat fungal infections by vaccines and immunotherapeutics to PNAG.

Staphylococcus aureus corneal infections: effect of the Panton-Valentine leukocidin (PVL) and antibody to PVL on virulence and pathology.

PURPOSE: Community-associated methicillin-resistant Staphylococcus aureus strains expressing Panton-Valentine leukocidin (PVL) are associated with severe skin and soft tissue infections, necrotizing pneumonia, and eye infections. We determined PVL's toxicity on infected mouse and cultured human corneal epithelial cells and the role of PVL and antibody to PVL in pathogenesis of murine keratitis. METHODS: Cytotoxicity on corneas and corneal epithelial cells was evaluated by LDH assays. Scratched corneas of female A/J mice were inoculated with approximately 107 CFU/eye of either WT S. aureus, isogenic ΔPVL, or strains overproducing PVL. Antibodies to PVL or control sera were topically applied to infected corneas 0, 24, and 32 hours postinfection, corneas scored for pathology and tissue levels of S. aureus were determined. RESULTS: PVL expression augmented the cytotoxicity of S. aureus on infected mouse corneas and human cultured corneal epithelial cells. Variable effects on leukocyte recruitment, pathogenesis, and immunity were obtained in the in vivo studies. Inactivation of PVL in USA300 strains caused reduced pathology and bacterial counts. Results were variable when comparing WT and ΔPVL USA400 strains, while USA400 strains overproducing PVL caused increased bacterial burdens. Topical treatment with polyclonal antibody to PVL yielded significant reductions in corneal pathology and bacterial CFU in corneas infected with USA300 strains, whereas effects were inconsistent in eyes infected with USA400 strains. CONCLUSIONS: PVL enhanced the virulence of a subset of MRSA strains in a keratitis model. Coupled with a variable effect of antibody treatment, it appears that PVL plays an inconsistent role in pathogenesis and immunity to S. aureus corneal infection.

Role of neutrophils, MyD88-mediated neutrophil recruitment, and complement in antibody-mediated defense against Pseudomonas aeruginosa keratitis.

Purpose. Ulcerative keratitis due to Pseudomonas aeruginosa is a sight-threatening disease leading to loss of vision due to corneal inflammation. A human IgG1 monoclonal antibody (MAb F429) to the alginate capsule significantly reduces pathology and bacterial burdens in the cornea when applied topically starting 8 hours post-infection. The purpose of this study was to determine whether local polymorphonuclear neutrophils (PMN) recruitment and complement were important lipopolysaccharide co-factors in MAb F429-mediated reductions in P. aeruginosa tissue levels and corneal pathology. Methods. MyD88 knock-out mice unable to recruit PMN to tissues, mice depleted of PMNs, or mice depleted of complement component C3 were topically treated with MAb F429 starting 8 hours post-infection and evaluated for bacterial levels and corneal pathology 48 hours after infection with two P. aeruginosa isolates. Results. An inability to recruit PMN or systemic PMN depletion plus topical application of MAb F429 resulted in less pathology in the eye, but bacterial burdens were markedly increased in the cornea, brains, and spleens of these mice, indicative of systemic spread. Intraperitoneal injection of cobra venom factor (CVF) reduced C3 levels in the cornea approximately 40%, which did not change the beneficial effects of MAb F429. Both systemic injection and topical application of CVF reduced local C3 levels >60%, which eliminated MAb-mediated reductions in corneal pathology and bacterial levels. Conclusions. PMN recruitment and complement are both needed for maximal in vivo efficacy of MAb F429 in therapeutically treating P. aeruginosa keratitis, and attempts to reduce pathology by limiting PMN influx could have consequences leading to more extensive local and systemic infection.