Disulfiram-Mitigating Unintended Effects.

Lyme disease caused by infection with a multitude of vector-borne organisms can sometimes be successfully treated in its very early stages. However, if diagnosis is delayed, this infection can become disseminated and, like another spirochetal infection syphilis, can affect multiple organ systems in the body, causing a wide variety of life-altering symptoms. Conventional antibiotic therapy may not be effective in eradicating the symptoms of the disease we know as Lyme disease. The recent literature has suggested that disulfiram (DSM) may be a potent drug in the armamentarium of physicians who treat chronic Lyme disease. The use of disulfiram in the treatment of Lyme disease started with a researcher who determined that DSM is bactericidal to spirochete. Encouraged by published case reports of apparent recovery from chronic Lyme disease, having prescribed DSM ourselves in the past for alcoholics who had a desire to stop drinking and prescribing it now for patients with chronic Lyme disease, we observed both predictable and potentially avoidable side effects not necessarily related to the ingestion of alcohol. We reviewed the published literature in PubMed and Google Scholar, using the following key words: Lyme Disease; Borrelia burgdorferi treatment; and disulfiram toxicity. This paper outlines the results of that research to help avoid some of the pitfalls inherent in this novel use of an old and established medication in the practice of clinical medicine.

Animal and human antibodies to distinct Staphylococcus aureus antigens mutually neutralize opsonic killing and protection in mice.

New prophylactic approaches are needed to control infection with the Gram-positive bacterium Staphylococcus aureus, which is a major cause of nosocomial and community-acquired infections. To develop these, greater understanding of protective immunity against S. aureus infection is needed. Human immunity to extracellular Gram-positive bacterial pathogens is primarily mediated by opsonic killing (OPK) via antibodies specific for surface polysaccharides. S. aureus expresses two such antigens, capsular polysaccharide (CP) and poly-N-acetyl glucosamine (PNAG). Here, we have shown that immunization-induced polyclonal animal antisera and monoclonal antibodies specific for either CP or PNAG antigens have excellent in vitro OPK activity in human blood but that when mixed together they show potent interference in OPK activity. In addition, reductions in antibody binding to the bacterial surface, complement deposition, and passive protection were seen in two mouse models of S. aureus infection. Electron microscopy, isothermal calorimetry, and surface plasmon resonance indicated that antibodies to CP and PNAG bound together via an apparent idiotype-anti-idiotype interaction. This interaction was also found in sera from humans with S. aureus bacteremia. These findings suggest that the lack of effective immunity to S. aureus infections in humans could be due, in part, to interference in OPK when antibodies to CP and PNAG antigens are both present. This information could be used to better design S. aureus vaccine components.

Evaluation of flagella and flagellin of Pseudomonas aeruginosa as vaccines.

Pseudomonas aeruginosa is a serious pathogen in hospitalized, immunocompromised, and cystic fibrosis (CF) patients. P. aeruginosa is motile via a single polar flagellum made of polymerized flagellin proteins differentiated into two major serotypes: a and b. Antibodies to flagella delay onset of infection in CF patients, but whether immunity to polymeric flagella and that to monomeric flagellin are comparable has not been addressed, nor has the question of whether such antibodies might negatively impact Toll-like receptor 5 (TLR5) activation, an important component of innate immunity to P. aeruginosa. We compared immunization with flagella and that with flagellin for in vitro effects on motility, opsonic killing, and protective efficacy using a mouse pneumonia model. Antibodies to flagella were superior to antibodies to flagellin at inhibiting motility, promoting opsonic killing, and mediating protection against P. aeruginosa pneumonia in mice. Protection against the flagellar type strains PAK and PA01 was maximal, but it was only marginal against motile clinical isolates from flagellum-immunized CF patients who nonetheless became colonized with P. aeruginosa. Purified flagellin was a more potent activator of TLR5 than were flagella and also elicited higher TLR5-neutralizing antibodies than did immunization with flagella. Antibody to type a but not type b flagella or flagellin inhibited TLR5 activation by whole bacterial cells. Overall, intact flagella appear to be superior for generating immunity to P. aeruginosa, and flagellin monomers might induce antibodies capable of neutralizing innate immunity due to TLR5 activation, but solid immunity to P. aeruginosa based on flagellar antigens may require additional components beyond type a and type b proteins from prototype strains.

Synthetic {beta}-(1->6)-linked N-acetylated and nonacetylated oligoglucosamines used to produce conjugate vaccines for bacterial pathogens.

Vaccines for pathogens usually target strain-specific surface antigens or toxins, and rarely is there broad antigenic specificity extending across multiple species. Protective antibodies for bacteria are usually specific for surface or capsular antigens. beta-(1-->6)-Poly-N-acetyl-d-glucosamine (PNAG) is a surface polysaccharide produced by many pathogens, including Staphylococcus aureus, Escherichia coli, Yersinia pestis, Bordetella pertussis, Acinetobacter baumannii, and others. Protective antibodies to PNAG are elicited when a deacetylated glycoform (deacetylated PNAG [dPNAG]; <30% acetate) is used in conjugate vaccines, whereas highly acetylated PNAG does not induce such antibodies. Chemical derivation of dPNAG from native PNAG is imprecise, so we synthesized both beta-(1-->6)-d-glucosamine (GlcNH(2)) and beta-(1-->6)-d-N-acetylglucosamine (GlcNAc) oligosaccharides with linkers on the reducing termini that could be activated to produce sulfhydryl groups for conjugation to bromoacetyl groups introduced onto carrier proteins. Synthetic 5-mer GlcNH(2) (5GlcNH(2)) or 9GlcNH(2) conjugated to tetanus toxoid (TT) elicited mouse antibodies that mediated opsonic killing of multiple S. aureus strains, while the antibodies that were produced in response to 5GlcNAc- or 9GlcNAc-TT did not mediate opsonic killing. Rabbit antibodies to 9GlcNH(2)-TT bound to PNAG and dPNAG antigens, mediated killing of S. aureus and E. coli, and protected against S. aureus skin abscesses and lethal E. coli peritonitis. Chemical synthesis of a series of oligoglucosamine ligands with defined differences in N acetylation allowed us to identify a conjugate vaccine formulation that generated protective immune responses to two of the most challenging bacterial pathogens. This vaccine could potentially be used to engender protective immunity to the broad range of pathogens that produce surface PNAG.

Protection against Escherichia coli infection by antibody to the Staphylococcus aureus poly-N-acetylglucosamine surface polysaccharide.

Poly-N-acetylglucosamine (PNAG) is a surface polysaccharide produced by Staphylococcus aureus and Staphylococcus epidermidis and is an effective target for opsonic and protective Ab for these two organisms. Recently, it has been found that Escherichia coli produces an exo-polysaccharide, designated polyglucosamine, that is biochemically indistinguishable from PNAG. We analyzed 30 E. coli strains isolated from urinary tract and neonatal bloodstream infections for the pga locus, PNAG antigen production, and susceptibility to opsonic killing and protection from lethal infection by Ab to PNAG. Twenty-six of 30 strains carried the pga locus, 25 of 30 expressed immunologically detectable PNAG, and 21 of 30 could be killed by rabbit IgG specific for the deacetylated form of the staphylococcal PNAG. Ab to staphylococcal PNAG protected mice against lethality from five different E. coli strains expressing PNAG. PNAG expression by both Gram-negative and Gram-positive organisms could make this antigen a conserved vaccine target for multiple pathogenic species of bacteria.

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.

Human monoclonal antibodies to Pseudomonas aeruginosa alginate that protect against infection by both mucoid and nonmucoid strains.

Two fully human mAbs specific for epitopes dependent on intact carboxylate groups on the C6 carbon of the mannuronic acid components of Pseudomonas aeruginosa alginate were found to promote phagocytic killing of both mucoid and nonmucoid strains as well as protection against both types of strains in a mouse model of acute pneumonia. The specificity of the mAbs for alginate was determined by ELISA and killing assays. Some strains of P. aeruginosa did not make detectable alginate in vitro, but in vivo protection against lethal pneumonia was obtained and shown to be due to rapid induction of expression of alginate in the murine lung. No protection against strains genetically unable to make alginate was achieved. These mAbs have potential to be passive therapeutic reagents for all strains of P. aeruginosa and the results document that alginate is a target for the proper type of protective Ab even when expressed at low levels on phenotypically nonmucoid strains.

Construction and characterization of a Pseudomonas aeruginosa mucoid exopolysaccharide-alginate conjugate vaccine.

Deterioration of lung function in patients with cystic fibrosis (CF) is closely associated with chronic pulmonary infection with mucoid Pseudomonas aeruginosa. The mucoid exopolysaccharide (MEP) from P. aeruginosa has been shown to induce opsonic antibodies in mice that are protective against this chronic infection. MEP-specific opsonic antibodies are also commonly found in the sera of older CF patients lacking detectable P. aeruginosa infection. When used in a human vaccine trial, however, MEP only minimally induced opsonic antibodies. To evaluate whether conjugation of MEP to a carrier protein could improve its immunogenicity, we bound thiolated MEP to keyhole limpet hemocyanin (KLH) by using succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as a linker. In contrast to the native MEP polymer, the MEP-KLH conjugate vaccine induced high titers of MEP-specific immunoglobulin G (IgG) in C3H-HeN mice and in a rabbit. Sera from mice immunized with MEP-KLH conjugate, but not from animals immunized with comparable doses of native MEP, demonstrated opsonic killing activity. Vaccination with MEP-KLH conjugate induced opsonic antibodies broadly cross-reactive to heterologous mucoid strains of P. aeruginosa. Preexisting nonopsonic antibodies to MEP are found in normal human sera, including young CF patients, and their presence impedes the induction of opsonic antibodies. Induction of nonopsonic antibodies by either intraperitoneal injection of MEP or injection or feeding of the cross-reactive antigen, seaweed alginate, reduced the level of overall IgG elicited by follow-up immunization with the MEP-KLH conjugate. However, the opsonic activity was lower only in the sera of MEP-KLH conjugate-immunized mice with preexisting antibodies induced by MEP but not with antibodies induced by seaweed alginate. Immunization with MEP-KLH elicited a significant proportion of antibodies specific to epitopes involving O-acetate residues, and this subpopulation of antibodies mediated opsonic killing of mucoid P. aeruginosa in vitro. These results indicate that conjugation of MEP to KLH significantly enhances its immunogenicity and the elicitation of opsonic antibodies in mice and rabbits, that the conjugate induces opsonic antibodies in the presence of preexisting nonopsonic antibodies, and that opsonic antibodies to MEP are directed at epitopes that include acetate residues on the uronic acid polymer.

Construction and characterization of a live, attenuated aroA deletion mutant of Pseudomonas aeruginosa as a candidate intranasal vaccine.

Antibodies to the lipopolysaccharide O antigen of Pseudomonas aeruginosa mediate high-level immunity, but protective epitopes have proven to be poorly immunogenic, while nonprotective or minimally protective O-antigen epitopes often elicit the best immune responses. With the goal of developing a broadly protective P. aeruginosa vaccine, we used a gene replacement system based on the Flp recombinase to construct an unmarked aroA deletion mutant of the P. aeruginosa serogroup O2/O5 strain PAO1. The resultant aroA deletion mutant of PAO1 is designated PAO1 Delta aroA. The aroA deletion was confirmed by both PCR and failure of the mutant to grow on minimal media lacking aromatic amino acids. When evaluated for safety and immunogenicity in mice, PAO1 Delta aroA could be applied either intranasally or intraperitoneally at doses up to 5 x 10(9) CFU per mouse without adverse effects. No dissemination of PAO1 Delta aroA to blood, liver, or spleen was detected after intranasal application, and histological evidence of pneumonia was minimal. Intranasal immunization of mice and rabbits elicited high titers of immunoglobulin G to whole bacterial cells and to heat-stable bacterial antigens of all seven prototypic P. aeruginosa serogroup O2/O5 strains. The mouse antisera mediated potent phagocytic killing of most of the prototypic serogroup O2/O5 strains, while the rabbit antisera mediated phagocytic killing of several serogroup-heterologous strains in addition to killing all O2/O5 strains. This live, attenuated P. aeruginosa strain PAO1 Delta aroA appears to be safe for potential use as an intranasal vaccine and elicits high titers of opsonic antibodies against multiple strains of the P. aeruginosa O2/O5 serogroup.