A novel tetrameric gp350 1-470 as a potential Epstein-Barr virus vaccine.

Infectious mononucleosis and B-cell transformation in response to infection with Epstein-Barr virus (EBV) is dependent upon binding of the EBV envelope glycoprotein gp350 to CD21 on B-cells. Gp350-specific antibody comprises most of the EBV neutralizing activity in the serum of infected patients, making this protein a promising target antigen for a prophylactic EBV vaccine. We describe a novel, tetrameric gp350-based vaccine that exhibits markedly enhanced immunogenicity relative to its monomeric counterpart. Plasmid DNA was constructed for synthesis, within transfected CHO cells, of a tetrameric, truncated (a.a. 1-470) gp350 protein (gp350(1-470)). Tetrameric gp350(1-470) induced ≈ 20-fold higher serum titers of gp350(1-470)-specific IgG and >19-fold enhancements in neutralizing titers at the highest dose, and was >25-fold more immunogenic on a per-weight basis than monomeric gp350(1-470). Further, epidermal immunization with plasmid DNA encoding gp350(1-470) tetramer induced 8-fold higher serum titers of gp350(1-470)-specific IgG relative to monomer. Tetrameric gp350(1-470) binding to human CD21 was >24-fold more efficient on a per-weight basis than monomer, but neither tetramer nor monomer mediated polyclonal human B-cell activation. Finally, the introduction of strong, universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes into the tetrameric gp350(1-470) had no effect on the gp350(1-470)-specific IgG response in naïve mice, and resulted in suppressed gp350(1-470)-specific IgG responses in TT-primed mice. Collectively, these data suggest that tetrameric gp350(1-470) is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.

Novel synthetic (poly)glycerolphosphate-based antistaphylococcal conjugate vaccine.

Staphylococcal infections are a major source of global morbidity and mortality. Currently there exists no antistaphylococcal vaccine in clinical use. Previous animal studies suggested a possible role for purified lipoteichoic acid as a vaccine target for eliciting protective IgG to several Gram-positive pathogens. Since the highly conserved (poly)glycerolphosphate backbone of lipoteichoic acid is a major antigenic target of the humoral immune system during staphylococcal infections, we developed a synthetic method for producing glycerol phosphoramidites to create a covalent 10-mer of (poly)glycerolphosphate for potential use in a conjugate vaccine. We initially demonstrated that intact Staphylococcus aureus elicits murine CD4(+) T cell-dependent (poly)glycerolphosphate-specific IgM and IgG responses in vivo. Naive mice immunized with a covalent conjugate of (poly)glycerolphosphate and tetanus toxoid in alum plus CpG-oligodeoxynucleotides produced high secondary titers of serum (poly)glycerolphosphate-specific IgG. Sera from immunized mice enhanced opsonophagocytic killing of live Staphylococcus aureus in vitro. Mice actively immunized with the (poly)glycerolphosphate conjugate vaccine showed rapid clearance of staphylococcal bacteremia in vivo relative to mice similarly immunized with an irrelevant conjugate vaccine. In contrast to purified, natural lipoteichoic acid, the (poly)glycerolphosphate conjugate vaccine itself exhibited no detectable inflammatory activity. These data suggest that a synthetic (poly)glycerolphosphate-based conjugate vaccine will contribute to active protection against extracellular Gram-positive pathogens expressing this highly conserved backbone structure in their membrane-associated lipoteichoic acid.

A randomized study of a monoclonal antibody (pagibaximab) to prevent staphylococcal sepsis.

BACKGROUND: Pagibaximab, a human chimeric monoclonal antibody developed against lipoteichoic acid, was effective against staphylococci preclinically and seemed safe and well tolerated in phase 1 studies. OBJECTIVE: To evaluate the clinical activity, pharmacokinetics, safety, and tolerability of weekly pagibaximab versus placebo infusions in very low birth weight neonates. PATIENTS AND METHODS: A phase 2, randomized, double-blind, placebo-controlled study was conducted at 10 NICUs. Patients with a birth weight of 700 to 1300 g and 2 to 5 days old were randomly assigned to receive 3 once-a-week pagibaximab (90 or 60 mg/kg) or placebo infusions. Blood was collected for pharmacokinetics, bacterial killing, and safety analyses. Adverse event and clinical outcome data were collected. RESULTS: Eighty-eight patients received pagibaximab at 90 (n = 22) or 60 (n = 20) mg/kg or placebo (n = 46). Groups were not different in demography, mortality, or morbidity. Pagibaximab demonstrated linear pharmacokinetics, a 14.5-day half-life, and nonimmunogenicity. Definite staphylococcal sepsis occurred in 0%, 20%, and 13% (P < .11) and nonstaphylococcal sepsis occurred in 0%, 10%, and 15% (P < .15) of patients in the 90 mg/kg, 60 mg/kg, and placebo groups, respectively. In all patients with staphylococcal sepsis, estimated or observed pagibaximab levels were <500 µg/mL (target level) at infection. CONCLUSIONS: Three once-a-week 90 or 60 mg/kg pagibaximab infusions, in high-risk neonates, seemed safe and well tolerated. No staphylococcal sepsis occurred in infants who received 90 mg/kg. Target levels were only consistently achieved after 2 to 3 doses. Dose optimization should enhance protection.

Phase 1/2 double-blind, placebo-controlled, dose escalation, safety, and pharmacokinetic study of pagibaximab (BSYX-A110), an antistaphylococcal monoclonal antibody for the prevention of staphylococcal bloodstream infections, in very-low-birth-weight neonates.

Staphylococcal sepsis is a major cause of morbidity and mortality in very-low-birth-weight (VLBW) infants. A human chimeric monoclonal antibody, pagibaximab, was developed against staphylococcal lipoteichoic acid. We evaluated the safety, tolerability, and pharmacokinetics of pagibaximab in VLBW neonates. A phase 1/2, randomized, double-blind, placebo-controlled, dose escalation study was conducted in VLBW infants (700 to 1,300 g) 3 to 7 days old. Patients received two doses 14 days apart of intravenous pagibaximab (10, 30, 60, or 90 mg/kg of body weight) or placebo in a 2:1 ratio. Blood and urine samples were obtained pre- and postinfusion for analysis of safety and pharmacokinetics, and data on adverse events were gathered. Staphylococcal organisms causing sepsis were collected and evaluated. Fifty-three patients received at least one dose of pagibaximab or placebo. The average gestational age was 27.6 weeks; the average birth weight was 1,003 g. All serious adverse events were deemed unrelated or probably not drug related. Morbidity and mortality were similar across treatment groups. No evidence of immunogenicity of pagibaximab was detected. Pagibaximab pharmacokinetics was linear. The mean clearance (CL), volume of distribution, and elimination half-life of pagibaximab were independent of dose. The serum half-life was 20.5 +/- 6.8 days. Pagibaximab enhanced serum opsonophagocytic activity. All staphylococci causing sepsis were opsonizable by pagibaximab. Two infusions of pagibaximab, administered 2 weeks apart to high-risk neonates appeared safe and tolerable, and pharmacokinetics were linear. Evaluation of more frequent doses, at the highest doses tested, in neonates at high-risk of staphylococcal sepsis, is warranted.

Lysostaphin eradicates established Staphylococcus aureus biofilms in jugular vein catheterized mice.

OBJECTIVES: Staphylococcus aureus infections associated with indwelling devices can be very difficult to treat due to the recalcitrant nature of bacterial biofilms to conventional antibiotics. Lysostaphin has been shown to clear S. aureus biofilms in vitro, and in this study we determined whether lysostaphin could also eradicate established S. aureus biofilms on implanted jugular vein catheters in mice. METHODS: Jugular vein catheterized mice (four to six per group) challenged with S. aureus developed multiorgan infection and biofilm infections on the catheters. The infected mice with established biofilms received various doses of recombinant lysostaphin through the catheters, administered up to three times daily for up to 4 days. Some mice also received lysostaphin combined with nafcillin. Following treatment, mice were sacrificed and cfu on the catheter and in the liver and heart were determined. In another set of experiments, implanted jugular vein catheters in mice were pre-instilled with lysostaphin to determine whether this pre-treatment would protect the mice from biofilm infection. RESULTS AND CONCLUSIONS: Lysostaphin administered at 15 mg/kg in combination with 50 mg/kg nafcillin three times per day for 4 days eradicated established S. aureus, including methicillin-resistant S. aureus, biofilms from implanted catheters and sterilized heart and liver infections of S. aureus-infected mice. Furthermore, a single pre-instillation of 10 mg/kg lysostaphin in catheters completely protected catheterized mice from a subsequent biofilm infection. These results demonstrate that lysostaphin is an effective treatment as well as prophylaxis for S. aureus biofilms on indwelling catheters.

Safety and pharmacokinetics of a chimerized anti-lipoteichoic acid monoclonal antibody in healthy adults.

A chimerized (murine/human) monoclonal antibody (pagibaximab) against lipoteichoic acid (LTA) and protective in animal models for coagulase-negative staphylococci (CONS) and Staphylococcus aureus bacteremia, was developed for prevention of staphylococcal infection in high-risk populations. This open label two-dose study of a single intravenous dose of 3 or 10 mg/kg of pagibaximab evaluated the safety/tolerability, pharmacokinetics, and opsonophagocytic activity of pagibaximab in healthy adults. Eight participants were enrolled (four in each dose group). No infusion, drug, or dose related adverse events occurred. Serum anti-LTA levels were dose-related; mean concentrations peaked at 87.75 and 259.24 microg/mL for 3 and 10 mg/kg groups, respectively. The half-life (beta) of pagibaximab was approximately 33 days. Opsonophagocytic activity of serum samples on a human clinical isolate of Staphylococcus epidermidis in a standard bacterial killing assay was dose-related, and peaked at a mean of 88.5 and 95.5% at 1:90 dilution for 3 and 10 mg/kg groups, respectively. Serum anti-LTA and opsonophagocytic activity levels exhibited statistically significant correlation. The results suggest that pagibaximab at 3 and 10 mg/kg administered as a single intravenous dose in healthy adults appears to: 1) provide preliminary safety and tolerability data, 2) produce dose-related serum anti-LTA and opsonophagocytic activity levels, 3) have a half-life similar to other immunoglobulin G1 antibodies, 4) exhibit statistically significant correlation between serum anti-LTA and opsonophagocytic activity levels. This study supports conducting safety and pharmacokinetic trials of pagibaximab in populations at high-risk of developing CONS infection.

The Staphylococcus aureus surface protein IsdA mediates resistance to innate defenses of human skin.

Resistance to human skin innate defenses is crucial for survival and carriage of Staphylococcus aureus, a common cutaneous pathogen and nasal colonizer. Free fatty acids extracted from human skin sebum possess potent antimicrobial activity against S. aureus. The mechanisms by which S. aureus overcomes this host defense during colonization remain unknown. Here, we show that S. aureus IsdA, a surface protein produced in response to the host, decreases bacterial cellular hydrophobicity rendering them resistant to bactericidal human skin fatty acids and peptides. IsdA is required for survival of S. aureus on live human skin. Reciprocally, skin fatty acids prevent the production of virulence determinants and the induction of antibiotic resistance in S. aureus and other Gram-positive pathogens. A purified human skin fatty acid was effective in treating systemic and topical infections of S. aureus suggesting that our natural defense mechanisms can be exploited to combat drug-resistant pathogens.

Lysostaphin as a treatment for systemic Staphylococcus aureus infection in a mouse model.

OBJECTIVES: With the isolation of clinical strains of Staphylococcus aureus carrying the gene that confers vancomycin resistance, the need for novel antistaphylococcals has become more urgent. Lysostaphin, an example of such a novel therapeutic, is an endopeptidase that rapidly lyses S. aureus through proteolysis of the staphylococcal cell wall. We evaluated its efficacy as a therapeutic agent for treatment of systemic S. aureus infection in a mouse model. METHODS: Mice (5-10 per group) challenged with methicillin-susceptible S. aureus developed bacteraemia and organ infections while mice challenged with methicillin-resistant S. aureus (MRSA) developed organ infections. The challenged mice received various intravenous doses of recombinant lysostaphin, administered once a day for 1-3 days when compared with treatment with oxacillin or vancomycin. Some mice also received treatment of lysostaphin combined with oxacillin or vancomycin. Following treatment, bacteraemia was determined, and mice were sacrificed and organ infection was determined. RESULTS AND CONCLUSIONS: Lysostaphin administered at 5 mg/kg once a day for 3 days consistently cleared S. aureus from the blood and the organs of infected mice. Furthermore, the combination of lysostaphin and oxacillin or vancomycin demonstrated increased efficacy against MRSA over lysostaphin alone allowing the therapeutic dose of lysostaphin to be reduced to 1 mg/kg. These results demonstrate that lysostaphin is an effective treatment for eradicating S. aureus from the blood and from the organs of infected mice.

Characterization of IsaA and SceD, two putative lytic transglycosylases of Staphylococcus aureus.

Bacterial cell wall peptidoglycan is a dynamic structure requiring hydrolysis to allow cell wall growth and division. Staphylococcus aureus has many known and putative peptidoglycan hydrolases, including two likely lytic transglycosylases. These two proteins, IsaA and SceD, were both found to have autolytic activity. Regulatory studies showed that the isaA and sceD genes are partially mutually compensatory and that the production of SceD is upregulated in an isaA mutant. The expression of sceD is also greatly upregulated by the presence of NaCl. Several regulators of isaA and sceD expression were identified. Inactivation of sceD resulted in impaired cell separation, as shown by light microscopy, and "clumping" of bacterial cultures. An isaA sceD mutant is attenuated for virulence, while SceD is essential for nasal colonization in cotton rats, thus demonstrating the importance of cell wall dynamics in host-pathogen interactions.

Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus.

Oxidative-stress resistance in Staphylococcus aureus is linked to metal ion homeostasis via several interacting regulators. In particular, PerR controls the expression of a regulon of genes, many of which encode antioxidants. Two PerR regulon members, ahpC (alkylhydroperoxide reductase) and katA (catalase), show compensatory regulation, with independent and linked functions. An ahpC mutation leads to increased H2O2 resistance due to greater katA expression via relief of PerR repression. Moreover, AhpC provides residual catalase activity present in a katA mutant. Mutation of both katA and ahpC leads to a severe growth defect under aerobic conditions in defined media (attributable to lack of catalase activity). This results in the inability to scavenge exogenous or endogenously produced H2O2, resulting in accumulation of H2O2 in the medium. This leads to DNA damage, the likely cause of the growth defect. Surprisingly, the katA ahpC mutant is not attenuated in two independent models of infection, which implies reduced oxygen availability during infection. In contrast, both AhpC and KatA are required for environmental persistence (desiccation) and nasal colonization. Thus, oxidative-stress resistance is an important factor in the ability of S. aureus to persist in the hospital environment and so contribute to the spread of human disease.

Identification of antigenic components of Staphylococcus epidermidis expressed during human infection.

A spectrum of in vivo-expressed Staphylococcus epidermidis antigens was identified by probing a bacteriophage lambda library of S. epidermidis genomic DNA with human serum from infected and uninfected individuals. This analysis resulted in identification of 53 antigen-encoding loci. Six antigenic polypeptides were expressed from these loci and purified. These polypeptides were the propeptide, mature amidase, and repeat sequence domains of the major autolysin AtlE, GehD (lipase), and two members of a conserved family of surface proteins (ScaA [AaE] and ScaB). AtlE, ScaA, and ScaB all exhibit human ligand binding capacity. Screening a bank of human serum samples revealed that there were significant increases in the amounts of reactive immunoglobulin G in infected individuals compared to the amounts in healthy individuals for the repeat sequence and mature amidase domains of AtlE, ScaB, and GehD. Vaccination of mice with recombinant antigens stimulated an immune response which in vitro opsonized S. epidermidis. In this study we identified prospective candidate antigens for prophylaxis or immunotherapy to control disease.

Identification of in vivo-expressed antigens of Staphylococcus aureus and their use in vaccinations for protection against nasal carriage.

A spectrum of in vivo-expressed Staphylococcus aureus antigens was identified by probing bacteriophage expression libraries of S. aureus with serum samples from infected and uninfected individuals. Eleven recombinant antigenic proteins were produced, and specific antibody titers in a large collection of human serum samples were determined. Significantly increased concentrations of reactive immunoglobulin G (IgG) to 7 antigens were found in serum samples from ill individuals, compared with those in healthy individuals. Significantly higher concentrations of reactive IgG to 4 antigens, including iron-responsive surface determinant (Isd) A and IsdH, were found in serum samples from healthy individuals who were not nasal carriers of S. aureus, compared with those in healthy carriers. Vaccination of cotton rats with IsdA or IsdH protected against nasal carriage. Also, IsdA is involved in adherence of S. aureus to human desquamated nasal epithelial cells and is required for nasal colonization in the cotton rat model. Thus, vaccination with these antigens may prevent S. aureus carriage and reduce the prevalence of human disease.

Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections.

Colonization of the anterior nares in approximately 37% of the population is a major risk factor for severe Staphylococcus aureus infections. Here we show that wall teichoic acid (WTA), a surface-exposed staphylococcal polymer, is essential for nasal colonization and mediates interaction with human nasal epithelial cells. WTA-deficient mutants were impaired in their adherence to nasal cells, and were completely unable to colonize cotton rat nares. This study describes the first essential factor for S. aureus nasal colonization.

The critical DNA flanking sequences of a CpG oligodeoxynucleotide, but not the 6 base CpG motif, can be replaced with RNA without quantitative or qualitative changes in Toll-like receptor 9-mediated activity.

Double- and single-stranded oligodeoxynucleotides containing unmethylated cytosine-guanosine (CpG) dinucleotides (CpG-ODN) activate immune cells via TLR9. In this report we synthesized hybrid DNA-RNA molecules (HDR) in order to further explore the structure-immune function relationship of CpG-ODN in TLR9 signaling and the potential immunomodulatory properties of RNA. We demonstrate that replacement of the deoxyadenosine flanking sequences, critical for the immune activating properties of CpG-ODN, with a similar number of adenosines, although not guanosines, cytosines, or uracils, maintains complete immunostimulatory activity of the hybrid oligonucleotide in vitro, whereas a similar RNA replacement of even 1 base of the required unmethylated 6 base DNA motif (purine-purine-CpG-pyrimidine-pyrimidine) results in a complete loss of activity. Regardless of whether the critical flanking sequence was RNA or DNA there was no significant change in the quantitative or qualitative immune-stimulating activity, or TLR-specificity of the resulting sequences, thus underscoring the relatively permissive functional role of the flanking sequence, and the more specific role of the motif in mediating TLR9 signaling. These data further support a potential role for RNA in immunomodulation.

Immunoprotective activity and safety of a respiratory syncytial virus vaccine: mucosal delivery of fusion glycoprotein with a CpG oligodeoxynucleotide adjuvant.

CpG oligodeoxynucleotides (ODN) were identified that stimulated immunoglobulin production and cell proliferation in cotton rat cells in vitro. Three of these ODN were used as a mucosal adjuvant in the noses of cotton rats immunized via this route with respiratory syncytial virus fusion (F) protein. The CpG ODN markedly increased the cotton rat humoral neutralizing-antibody response to respiratory syncytial virus. Such immunized animals had a marked reduction in the production of infectious virus after a live-virus challenge. Animals immunized with the combination of F protein and CpG developed enhanced pulmonary pathology consisting of alveolitis and interstitial pneumonitis after a live-virus challenge. Similar enhanced disease has been seen in cotton rats and children immunized with formalin-inactivated respiratory syncytial virus.

Lysostaphin disrupts Staphylococcus aureus and Staphylococcus epidermidis biofilms on artificial surfaces.

Staphylococci often form biofilms, sessile communities of microcolonies encased in an extracellular matrix that adhere to biomedical implants or damaged tissue. Infections associated with biofilms are difficult to treat, and it is estimated that sessile bacteria in biofilms are 1,000 to 1,500 times more resistant to antibiotics than their planktonic counterparts. This antibiotic resistance of biofilms often leads to the failure of conventional antibiotic therapy and necessitates the removal of infected devices. Lysostaphin is a glycylglycine endopeptidase which specifically cleaves the pentaglycine cross bridges found in the staphylococcal peptidoglycan. Lysostaphin kills Staphylococcus aureus within minutes (MIC at which 90% of the strains are inhibited [MIC(90)], 0.001 to 0.064 microg/ml) and is also effective against Staphylococcus epidermidis at higher concentrations (MIC(90), 12.5 to 64 microg/ml). The activity of lysostaphin against staphylococci present in biofilms compared to those of other antibiotics was, however, never explored. Surprisingly, lysostaphin not only killed S. aureus in biofilms but also disrupted the extracellular matrix of S. aureus biofilms in vitro on plastic and glass surfaces at concentrations as low as 1 microg/ml. Scanning electron microscopy confirmed that lysostaphin eradicated both the sessile cells and the extracellular matrix of the biofilm. This disruption of S. aureus biofilms was specific for lysostaphin-sensitive S. aureus, as biofilms of lysostaphin-resistant S. aureus were not affected. High concentrations of oxacillin (400 microg/ml), vancomycin (800 microg/ml), and clindamycin (800 microg/ml) had no effect on the established S. aureus biofilms in this system, even after 24 h. Higher concentrations of lysostaphin also disrupted S. epidermidis biofilms.

Lysostaphin cream eradicates Staphylococcus aureus nasal colonization in a cotton rat model.

The anterior nares are a primary ecologic niche for Staphylococcus aureus, and nasal colonization by this opportunistic pathogen increases the risk of development of S. aureus infection. Clearance of S. aureus nasal colonization greatly reduces this risk. Mupirocin ointment is the current standard of care for clearance of S. aureus nasal colonization, but resistance to this antibiotic is emerging. Lysostaphin is a glycylglycine endopeptidase which specifically cleaves the cross-linking pentaglycine bridges in the cell walls of staphylococci. Lysostaphin is extremely staphylocidal (MIC at which 90% of isolates are inhibited, 0.001 to 0.064 micro g/ml) and rapidly lyses both actively growing and quiescent S. aureus. This study demonstrates that a single application of 0.5% lysostaphin (actual dose, approximately 150 micro g of lysostaphin), formulated in a petrolatum-based cream, dramatically reduces S. aureus nasal colonization in 100% of animals tested and eradicates S. aureus nasal colonization in 93% of animals in a cotton rat model. A single dose of lysostaphin cream is more effective than a single dose of mupirocin ointment in eradicating S. aureus nasal colonization in this animal model. The lantibiotic peptide nisin, which has potent in vitro antistaphylococcal activity, was ineffective in reducing staphylococcal nasal carriage in this model. Nasal colonization was not reduced after three treatments with 5% nisin ( approximately 1,500 micro g/dose) in any of the treated animals. Lysostaphin formulated in cream may prove to be a superior alternative to mupirocin ointment for clearance of S. aureus nasal colonization.

Proliferative assays for B cell function.

This unit describes procedures for measuring the capacity of purified B cells to undergo proliferation. The method centers on the use of polyclonal stimulating agents (mitogens) because these agents stimulate the majority of B cells and because the alternative (measurement of antigen-induced proliferation) requires the laborious procedures of isolating antigen-specific B cells (which are otherwise present in too low a concentration in whole B cell populations). Cross-linking of the B cell antigen receptor, surface immunoglobulin (sIg), by specific antigen stimulates cells to proliferate prior to secreting Ig. For this purpose, monoclonal or heterologous affinity-purified anti-Ig antibodies are used. B cells can also be stimulated to proliferate by antigen-nonspecific reagents (mitogens), and it is also critical to study the role of these mitogens in B cell responses. Both of these systems have the advantage that the majority of B cells will be activated. The first basic protocol describes B cell proliferation induced by two commonly used stimulants--anti-Ig antibody (either anti-IgM or anti-IgD) and lipopolysaccharide (LPS)--as measured by incorporation of [3H]thymidine into dividing cells. Alternate protocols describe other commonly used mitogens as well as other means of measuring cell proliferation.