Polysaccharide poly-N-acetylglucosamine Research Articles

Vaccination Against Poly-N-Acetylglucosamine Decreases Neutrophil Activation and Gvhd While Maintaining Microbial Diversity

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for different malignant and non-malignant hematologic diseases. Major life-threatening complications after allo-HCT are acute graft-versus-host disease (aGVHD) and severe infections. The incidence of GVHD after allo-HCT remains high, despite prophylactic immunosuppressive medication. According to the CIBMTR database, 60% of patients undergoing allo-HCT develop at least grade II-IV aGVHD. Microbial invasion into the intestinal mucosa after allo-HCT triggers the activation of neutrophil granulocytes (neutrophils) and requires antibiotic treatment to prevent sepsis. However, antibiotics lead to a loss of microbial diversity, which is connected to a higher incidence of aGVHD. Anti-microbial therapies which eliminate invading bacteria and diminish neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution for this could be the use of anti-microbial antibodies that target and mark invading pathogens resulting in their elimination by innate immune cells. Therefore we investigated the potency of both active and passive immunization against the conserved microbial surface polysaccharide Poly-N-acetylglucosamine (PNAG) which is expressed on various pathogens in a murine aGVHD model. We could detect reduced aGVHD related mortality in mice which underwent treatment with an antibody against PNAG (anti-PNAG) or were actively immunized against PNAG. Sequencing analysis of the microbiota before and after allo-HCT showed that anti-PNAG treatment did not have any effect luminal microbial diversity. Mechanistically, we could show that anti-PNAG antibody treatment caused more abundant neutrophil recruitment to the intestinal submucosa. This supports the concept that neutrophils are able to effectively eliminate any opsonized bacteria in this tissue, leading to reduced local inflammation. In agreement with this we observed lower intestinal inflammation and reduced myeloperoxidase activity as well as proliferation of neutrophils in the small intestine of mice treated with anti-PNAG antibody. We demonstrate the potency of targeting PNAG as a novel antimicrobial treatment option in aGVHD by reducing uncontrolled neutrophil activation and thereby interfering with aGVHD without affecting commensal intestinal microbial diversity. Anti-PNAG-antibodies enhance anti-microbial immunity, while reducing the activation of neutrophil-mediated downstream immunopathology which is a novel approach to reduce the severity of GVHD. This could be translated into a clinical application given the modest toxicity profile of vaccination and the availability of fully human anti-PNAG antibodies, both of which have been tested in phase 1 trials in humans (ClinicalTrials.gov Identifier: NCT02853617). Disclosures Cywes-Bentley: OneBiopharma, Inc.: Honoraria. Koenecke:Novartis: Other: none. Pier:OneBiopharma, Inc.: Equity Ownership, Honoraria; Alopexx Vaccine, LLC: Equity Ownership, Honoraria.

Immunization against poly-N-acetylglucosamine reduces neutrophil activation and GVHD while sparing microbial diversity

Microbial invasion into the intestinal mucosa after allogeneic hematopoietic cell transplantation (allo-HCT) triggers neutrophil activation and requires antibiotic interventions to prevent sepsis. However, antibiotics lead to a loss of microbiota diversity, which is connected to a higher incidence of acute graft-versus-host disease (aGVHD). Antimicrobial therapies that eliminate invading bacteria and reduce neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution would be the use of antimicrobial antibodies that target invading pathogens, ultimately leading to their elimination by innate immune cells. In a mouse model of aGVHD, we investigated the potency of active and passive immunization against the conserved microbial surface polysaccharide poly-N-acetylglucosamine (PNAG) that is expressed on numerous pathogens. Treatment with monoclonal or polyclonal antibodies to PNAG (anti-PNAG) or vaccination against PNAG reduced aGVHD-related mortality. Anti-PNAG treatment did not change the intestinal microbial diversity as determined by 16S ribosomal DNA sequencing. Anti-PNAG treatment reduced myeloperoxidase activation and proliferation of neutrophil granulocytes (neutrophils) in the ileum of mice developing GVHD. In vitro, anti-PNAG treatment showed high antimicrobial activity. The functional role of neutrophils was confirmed by using neutrophil-deficient LysMcreMcl1fl/fl mice that had no survival advantage under anti-PNAG treatment. In summary, the control of invading bacteria by anti-PNAG treatment could be a novel approach to reduce the uncontrolled neutrophil activation that promotes early GVHD and opens a new avenue to interfere with aGVHD without affecting commensal intestinal microbial diversity.

In vitro evaluation of complement deposition and opsonophagocytic killing of Rhodococcus equi mediated by poly-N-acetyl glucosamine hyperimmune plasma compared to commercial plasma products.

BackgroundThe bacterium Rhodococcus equi can cause severe pneumonia in foals. The absence of a licensed vaccine and limited effectiveness of commercial R. equi hyperimmune plasma (RE‐HIP) create a great need for improved prevention of this disease.HypothesisPlasma hyperimmune to the capsular polysaccharide poly‐N‐acetyl glucosamine (PNAG) would be significantly more effective than RE‐HIP at mediating complement deposition and opsonophagocytic killing (OPK) of R. equi.AnimalsVenipuncture was performed on 9 Quarter Horses.MethodsThe ability of the following plasma sources to mediate complement component 1 (C1) deposition onto either PNAG or R. equi was determined by ELISA: (1) PNAG hyperimmune plasma (PNAG‐HIP), (2) RE‐HIP, and (3) standard non‐hyperimmune commercial plasma (SP). For OPK, each plasma type was combined with R. equi, equine complement, and neutrophils isolated from horses (n = 9); after 4 hours, the number of R. equi in each well was determined by quantitative culture. Data were analyzed using linear mixed‐effects regression with significance set at P < .05.ResultsThe PNAG‐HIP and RE‐HIP were able to deposit significantly (P < .05) more complement onto their respective targets than the other plasmas. The mean proportional survival of R. equi opsonized with PNAG‐HIP was significantly (P < .05) less (14.7%) than that for SP (51.1%) or RE‐HIP (42.2%).Conclusions and Clinical ImportancePlasma hyperimmune to PNAG is superior to RE‐HIP for opsonizing and killing R. equi in vitro. Comparison of these 2 plasmas in field trials is warranted because of the reported incomplete effectiveness of RE‐HIP.

Antibodies to Conserved Surface Polysaccharides Protect Mice Against Bacterial Conjunctivitis.

PurposeBacterial 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.MethodsBacteria 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.ResultsAll 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.ConclusionsAntibodies 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.

Novel glycopolymer sensitizes Burkholderia cepacia complex isolates from cystic fibrosis patients to tobramycin and meropenem.

Burkholderia cepacia complex (Bcc) infection, associated with cystic fibrosis (CF) is intrinsically multidrug resistant to antibiotic treatment making eradication from the CF lung virtually impossible. Infection with Bcc leads to a rapid decline in lung function and is often a contraindication for lung transplant, significantly influencing morbidity and mortality associated with CF disease. Standard treatment frequently involves antibiotic combination therapy. However, no formal strategy has been adopted in clinical practice to guide successful eradication. A new class of direct-acting, large molecule polycationic glycopolymers, derivatives of a natural polysaccharide poly-N-acetyl-glucosamine (PAAG), are in development as an alternative to traditional antibiotic strategies. During treatment, PAAG rapidly targets the anionic structural composition of bacterial outer membranes. PAAG was observed to permeabilize bacterial membranes upon contact to facilitate potentiation of antibiotic activity. Three-dimensional checkerboard synergy analyses were used to test the susceptibility of eight Bcc strains (seven CF clinical isolates) to antibiotic combinations with PAAG or ceftazidime. Potentiation of tobramycin and meropenem activity was observed in combination with 8–128 μg/mL PAAG. Treatment with PAAG reduced the minimum inhibitory concentration (MIC) of tobramycin and meropenem below their clinical sensitivity breakpoints (≤4 μg/mL), demonstrating the ability of PAAG to sensitize antibiotic resistant Bcc clinical isolates. Fractional inhibitory concentration (FIC) calculations showed PAAG was able to significantly potentiate antibacterial synergy with these antibiotics toward all Bcc species tested. These preliminary studies suggest PAAG facilitates a broad synergistic activity that may result in more positive therapeutic outcomes and supports further development of safe, polycationic glycopolymers for inhaled combination antibiotic therapy, particularly for CF-associated Bcc infections.

Active and Passive Immunization Against Staphylococcus aureus Periprosthetic Osteomyelitis in Rats.

Staphylococcus aureus infection associated with orthopedic implants cannot always be controlled. We used a knee prosthesis model with implant-related osteomyelitis in rats to explore induction of an effective immune response with active and passive immunization. Fifty-two Sprague-Dawley rats were divided into active (N=28) and passive immunization groups (N=24). A bacterial inoculum of 103 S. aureus MN8 was injected into the tibia and the femur marrow before insertion of a non-constrained knee prosthesis in each rat. The active-immunization group received a synthetic oligosaccharide of polysaccharide poly-N-acetylglucosamine (PNAG), 9G1cNH2 and the passive-immunization group received immunization with immunoglobulin from rabbits infected with S. aureus. Active immunization against PNAG significantly reduced the consequences of osteomyelitis infection from PNAG-producing intercellular adhesion (ica+) but not ica- S. aureus. Passive immunization resulted in better clinical assessments in animals challenged with either ica+ or ica- S. aureus, suggesting a lack of specificity in this antiserum.

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.

Noninvasive Staphylococcus aureus biofilm determination in chronic rhinosinusitis by detecting the exopolysaccharide matrix component poly‐N‐acetylglucosamine

The role that bacterial biofilms might play in recalcitrant forms of chronic rhinosinusitis (CRS) is increasingly being recognized. However, the detection of bacteria existing in this form, using standard culture, is limited by their unique metabolically inactive properties. All current biofilm diagnostic modalities require invasive mucosal biopsies, which limit their use to the operating theatre. Twenty CRS patients and 5 controls were enrolled in a prospective study to assess the feasibility of noninvasively diagnosing S. aureus biofilms by detecting the biofilm matrix polysaccharide poly-N-acetylglucosamine (PNAG). An immunofluorescence protocol was developed for PNAG detection and compared with both standard microbiological cultures and fluorescence in situ hybridization (FISH). Thirteen of 20 CRS patients had evidence of S. aureus biofilm formation using FISH. Of these, 12 had detectable PNAG. Interestingly none of the S. aureus FISH-negative patients were PNAG-positive despite the presence of coagulase-negative Staphylococci biofilms, some of which may exhibit PNAG in their pathogenic forms. The development of a noninvasive S. aureus biofilm diagnostic test provides a reliable means to identify a high-risk group of CRS patients who harbor S. aureus biofilms. The ability to be used outside of the perioperative period to assess surgical efficacy, guide management, and evaluate new treatment modalities provides a significant advance in this field of research and clinical practice. This study has confirmed the feasibility of noninvasive detection of S. aureus biofilms with a simple test that produces results comparable to the more invasive methods that are currently relied upon.

The Role of Epitope Specificity in the Human Opsonic Antibody Response to the Staphylococcal Surface Polysaccharide Poly N‐Acetyl Glucosamine

The staphylococcal surface polysaccharide poly N-acetyl glucosamine (PNAG) is a target for killing and protective antibody in animals. We investigated the human antibody response and specificity of binding and opsonic antibodies for different epitopes on PNAG in serum samples from patients with cystic fibrosis (CF) colonized and not colonized with Staphylococcus aureus. Serum samples from patients with CF colonized and not colonized with S. aureus were used to compare levels and specificities of binding and opsonic antibodies to native PNAG (>95% acetylation) and deacetylated PNAG (dPNAG, approximately 15% acetylation). Colonized patients had higher killing activity mediated by opsonic antibody than did noncolonized patients in a PNAG-specific opsonophagocytic assay (P<.0001) but no difference in average levels of antibody to either PNAG or dPNAG by enzyme-linked immunosorbent assay. Killing activity in serum samples of the colonized patients correlated with the level of IgG specific to dPNAG more than to native PNAG. dPNAG and PNAG shared expression of the epitopes binding opsonic antibody, as evidenced by comparable inhibition of opsonophagocytic killing by both antigens. Affinity-purified antibodies specific to dPNAG were superior in mediating opsonic killing. Human antibodies to PNAG that mediate opsonic killing bind primarily to the nonacetylated epitopes of this antigen, which indicates that these antigenic determinants are the dominant targets of the functional human antibody response to staphylococcal PNAG.

Poly- N -Acetylglucosamine Production in Staphylococcus aureus Is Essential for Virulence in Murine Models of Systemic Infection

The contribution of the Staphylococcus aureus surface polysaccharide poly-N-acetylglucosamine (PNAG) to virulence was evaluated in three mouse models of systemic infection: bacteremia, renal abscess formation, and lethality following high-dose intraperitoneal (i.p.) infection. Deletion of the intercellular adhesin (ica) locus that encodes the biosynthetic enzymes for PNAG production in S. aureus strains Mn8, Newman, and NCTC 10833 resulted in mutant strains with significantly reduced abilities to maintain bacterial levels in blood following intravenous or i.p. injection, to spread systemically to the kidneys following i.p. injection, or to induce a moribund/lethal state following i.p. infection. In the bacteremia model, neither growth phase nor growth medium used to prepare the S. aureus inoculum affected the conclusion that PNAG production was needed for full virulence. As the SarA regulatory protein has been shown to affect ica transcription, PNAG synthesis, and biofilm formation, we also evaluated S. aureus strains Mn8 and 10833 deleted for the sarA gene in the renal infection model. A decrease in PNAG production was seen in sarA mutants using immunoblots of cell surface extracts but was insufficient to reduce the virulence of sarA-deleted strains in this model. S. aureus strains deleted for the ica genes were much more susceptible to antibody-independent opsonic killing involving human peripheral blood leukocytes and rabbit complement. Thus, PNAG confers on S. aureus resistance to killing mediated by these innate host immune mediators. Overall, PNAG production by S. aureus appears to be a critical virulence factor as assessed in murine models of systemic infection.

The teicoplanin-associated locus regulator (TcaR) and the intercellular adhesin locus regulator (IcaR) are transcriptional inhibitors of the ica locus in Staphylococcus aureus.

Infections involving Staphylococcus aureus are often more severe and difficult to treat when the organism assumes a biofilm mode of growth. The polysaccharide poly-N-acetylglucosamine (PNAG), also known as polysaccharide intercellular adhesin, is synthesized by the products of the intercellular adhesin (ica) locus and plays a key role in biofilm formation. Numerous conditions and exogenous factors influence ica transcription and PNAG synthesis, but the regulatory factors and pathways through which these environmental stimuli act have been only partially characterized. We developed a DNA affinity chromatography system to purify potential regulatory proteins that bind to the ica promoter region. Using this technique, we isolated four proteins, including the staphylococcal gene regulator SarA, a MarR family transcriptional regulator of the teicoplanin-associated locus TcaR, DNA-binding protein II, and topoisomerase IV, that bound to the ica promoter. Site-directed deletion mutagenesis of tcaR indicated that TcaR was a negative regulator of ica transcription, but deletion of tcaR alone did not induce any changes in PNAG production or in adherence to polystyrene. We also investigated the role of IcaR, encoded within the ica locus but divergently transcribed from the biosynthetic genes. As has been shown previously in Staphylococcus epidermidis, we found that IcaR was also a negative regulator of ica transcription in S. aureus. We also demonstrate that mutation of icaR augmented PNAG production and adherence to polystyrene. Transcription of the ica locus, PNAG production, and adherence to polystyrene were further increased in a tcaR icaR double mutant. In summary, TcaR appeared to be a weak negative regulator of transcription of the ica locus, whereas IcaR was a strong negative regulator, and in their absence PNAG production and biofilm formation were enhanced.