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In an effort to characterize important epitopes ofStaphylococcus aureusiron-regulated surface determinant B (IsdB), murine IsdB-specific monoclonal antibodies (MAbs) were isolated and characterized. A panel of 12 MAbs was isolated. All 12 MAbs recognized IsdB in enzyme-linked immunosorbent assays and Western blots; 10 recognized native IsdB expressed byS. aureus. The antigen epitope binding of eight of the MAbs was examined further. Three methods were used to assess binding diversity: MAb binding to IsdB muteins, pairwise binding to recombinant IsdB, and pairwise binding to IsdB-expressing bacteria. Data from these analyses indicated that MAbs could be grouped based on distinct or nonoverlapping epitope recognition. Also, MAb binding to recombinant IsdB required a significant portion of intact antigen, implying conformational epitope recognition. Four MAbs with nonoverlapping epitopes were evaluated for in vitro opsonophagocytic killing (OPK) activity and efficacy in murine challenge models. These were isotype switched from immunoglobulin G1 (IgG1) to IgG2b to potentially enhance activity; however, this isotype switch did not appear to enhance functional activity. MAb 2H2 exhibited OPK activity (≥50% killing in the in vitro OPK assay) and was protective in two lethal challenge models and a sublethal indwelling catheter model. MAb 13C7 did not exhibit OPK (

There is a clear unmet medical need for a vaccine that would prevent infections from Staphylococcus aureus (S. aureus). To validate antigens as potential vaccine targets it has to be demonstrated that the antigens are expressed in vivo. Using murine bacteremia and wound infection models, we demonstrate that the expression of clumping factor A (ClfA) and capsular polysaccharide antigens are heterogeneous and dependent on the challenge strains examined and the in vivo microenvironment. We also demonstrate opsonophagocitic activity mediated by either antigen is not impeded by the presence of the other antigen. The data presented in this report support a multiantigen approach for the development of a prophylactic S. aureus vaccine to ensure broad coverage against this versatile pathogen.

The outer membrane protein LP2086, a human factor H binding protein, is undergoing clinical trials as a vaccine against invasive serogroup B meningococcal (MnB) disease. As LP2086 is a surface protein, expression of capsular polysaccharide could potentially limit accessibility of anti-LP2086 antibodies to LP2086 expressed on the surface of bacteria. To determine whether variability in expression levels of the serogroup B capsule (Cap B) might interfere with accessibility of anti-LP2086 antibody binding to LP2086, we evaluated the ability of anti-Cap B and anti-LP2086 antibodies to bind to the surface of 1263 invasive clinical MnB strains by flow cytometry. One of the anti-LP2086 monoclonal antibodies used recognizes virtually all LP2086 sequence variants. Our results show no correlation between the amount of Cap B expressed and the binding of anti-LP2086 antibodies. Furthermore, the susceptibility of MnB bacteria to lysis by anti-LP2086 immune sera was independent of the level of Cap B expressed. The data presented in this paper demonstrates that Cap B does not interfere with the binding of antibodies to LP2086 expressed on the outer membrane of MnB clinical isolates.

Staphylococcus aureus is a clinically important capsule-forming bacterium. The capsule polysaccharide (CPs) occurs as different chemical structures depending on the serotype of the organism, but one form, capsular polysaccharide type 8 (CPs8) found in clinical isolates, is largely unstudied. The potential of CPs8 as a vaccine target was evaluated using two approaches. The first approach used a conjugate vaccine, made by chemically linking purified CPs8 to the outer membrane protein complex of N. meningitidis serotype B (OMPC). In efficacy studies, the CPs8-OMPC conjugate vaccine was immunogenic in Balb/c mice, however the immune response gave no protection from death after a lethal intravenous (IV) challenge with S. aureus Becker. In the second approach, two monoclonal antibodies were produced against CPs8 (mAbs 8E8 and 1C10). These were found to have functional activity in an opsonophagocytic killing assay (OPA), and provided protection from a lethal challenge when bacteria were pre-opsonized ex vivo before intra-peritoneal (IP) challenge. However, mAb 8E8 was not efficacious in the lethal challenge model, in which antibodies were passively transferred to the peritoneum and the animals were infected via the tail vein 18-24 h later. Additionally, the monoclonal antibodies did not opsonize capsule-expressing S. aureus Becker obtained from in vivo growth conditions. These results indicated that functional capsule antibodies may not be sufficient for protection from S. aureus under all in vivo conditions.

The combined power of genomics and proteomics has led to many advances in the discovery of bacterial vaccine targets. The 'Holy Grail' for a vaccine is to be pathogen specific yet conserved among all strains, so that universal coverage is possible with the minimal number of antigens. Genomics allows us to target conserved proteins, while proteomics tells us what is actually expressed and what is accessible to antibodies. Achievements using these latest approaches are exemplified by the vaccine clinical trials that are ongoing for protein targets against Neiserria meningitidis and Staphylococcus aureus along with promising discoveries that have been made for other pathogens including Streptococcus pneumoniae and Streptococcus pyogenes. These developments are discussed in this review.

Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for “near transporter”) present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S -transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants ( K D ) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel β-barrel with the strand order (-β1 ↓ -β2 ↑ -β3 ↓ -β6 ↑ -β5 ↓ -β4 ↑ -β7 ↓ -β8 ↑ ), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.

MntC is the metal-binding protein component of the Mn2+-specific MntABC transporter from the human pathogen Staphylococcus aureus. MntC orthologs are found in all Gram positive bacteria, the protein is attached to the bacterial membrane through a lipid anchor and captures Mn2+ ion from the environment with high affinity for subsequent transport across the transmembrane channel of the transporter. MntC consists of two homologous domains connected through a long ‘backbone’ α-helix, with a metal-binding site located at the interface of those domains. The long α-helix restricts domain mobility, making it harder for the bound ligand (Mn2+) to escape the binding site and, correspondingly, increasing the metal-binding affinity of the protein. The physiological role of Mn2+ binding by MntC in bacterial resistance to oxidative stress is well documented. MntC is also able to bind other divalent metals (Zn2+, Co2+, Ni2+, and Cd2+), although it is not yet clear whether this has any biological relevance. Binding of divalent metals to the protein can be reversible and irreversible, depending on the nature of the metal. X-ray data provide a plausible explanation for the two binding modes: the X-ray structure contains two molecules in the asymmetric unit, with the metal-binding site being solvent accessible in one molecule and inaccessible in the other. Serum antibodies to MntC (or orthologs of MntC) are detected following exposure to staphylococcal species in animal models as well as in human sera from patients with S. aureus infections. Immunization with a recombinant expressed MntC protein antigen induces protective immunity against S. aureus in preclinical models of infection. As such, MntC is a promising vaccine candidate and is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of staphylococcal infections. 3D Structure 3D structure of S. aureus MntC (PDB code 4K3V). The figure is rendered using Accelrys Discovery Studio Visualizer 2.5. The N-terminal domain of the protein is shown in red, the C-terminal domain is shown in green, and the connecting ‘backbone’ α-helix is shown in orange. The Mn2+ ion in the binding site is shaded in purple. Two orientations of the molecule are shown. Keywords: MntC; manganese; X-ray crystallography; metal binding; DSC; ITC; S. aureus; vaccine

Staphylococcus aureus is a versatile pathogen of medical significance, using multiple virulence factors to cause disease. A prophylactic S. aureus 4-antigen (SA4Ag) vaccine comprising capsular polysaccharide (types 5 and 8) conjugates, clumping factor A (ClfA) and manganese transporter C (MntC) is under development. This study was designed to characterize S. aureus isolates recovered from infected patients and also to investigate approaches for examining expression of S. aureus vaccine candidates and the host response during human infection. Confirmation of antigen expression in different disease states is important to support the inclusion of these antigens in a prophylactic vaccine. Hospitalized patients with diagnosed S. aureus wound (27) or bloodstream (24) infections were enrolled. Invasive and nasal carriage S. aureus isolates were recovered and characterized for genotypic diversity. S. aureus antigen expression was evaluated directly by real-time, quantitative, reverse-transcriptase PCR (qRT-PCR) analysis and indirectly by serology using a competitive Luminex immunoassay. Study isolates were genotypically diverse and all had the genes encoding the antigens present in the SA4Ag vaccine. S. aureus nasal carriage was detected in 55% of patients, and in those subjects 64% of the carriage isolates matched the invasive strain. In swab samples with detectable S. aureus triosephosphate isomerase housekeeping gene expression, RNA transcripts encoding the S. aureus virulence factors ClfA, MntC, and capsule polysaccharide were detected by qRT-PCR. Antigen expression was indirectly confirmed by increases in antibody titer during the course of infection from acute to convalescent phase. Demonstration of bacterial transcript expression together with immunological response to the SA4Ag antigens in a clinically relevant patient population provides support for inclusion of these antigens in a prophylactic vaccine.

Vaccination of children has had a major impact on the morbidity and mortality of many infectious diseases globally. However, with age, immune responses to vaccines can be less robust, which can be further enhanced by underlying diseases that are common in the older adult. In many countries around the globe booster vaccinations against diphtheria, tetanus, and pertussis are recommended for adults. For the older adult, vaccination against pneumococcal diseases, influenza and herpes zoster are also recommended. Despite these recommendations, the widespread use of these vaccines in the adult population clearly lags behind the vaccine uptake and successes documented for pediatric vaccination programs. Furthermore, extensive and sometimes inappropriate use of antibiotics have fostered the emergence of antibiotic-resistant bacteria (e.g., methicillin resistant Staphylococcus aureus (MRSA)) as well as increased susceptibility in the elderly to bacterial species such as Clostridium difficile. Infectious diseases remain an important unmet medical need and new concepts to successfully implement vaccination of adults are urgently needed.

Staphylococcus epidermidis is a significant cause of nosocomial disease. However, the taxonomy of this pathogen, particularly at subspecies level, is unclear. A multilocus sequence typing (MLST) scheme has therefore been investigated as a tool to elucidate taxonomic relationships within this group, based on genetic relatedness. DNA sequences for internal fragments of seven housekeeping genes were compared in 47 geographically and temporally diverse S. epidermidis isolates that were obtained from clinical infections. Twenty-three different allelic profiles were detected; 17 of these were represented by single strains and the largest profile group contained 17 isolates. Diversity of the same collection of isolates was investigated by using PFGE of SmaI-digested genomic DNA to test the discrimination and validity of the MLST approach. Isolates within the largest profile group were resolved into four distinct PFGE clusters on the basis of their SmaI digest patterns. Isolates within other profile groups that contained multiple isolates had matching PFGE SmaI patterns within each group. It appears that MLST is an effective method for grouping S. epidermidis strains at the subspecies level; however, it is not as discriminatory as it has been for other species for which MLST schemes have been established and, used alone, would not be a useful method for epidemiological studies. In addition, it was demonstrated that this method was effective for confirming the identity of S. epidermidis CoNS (coagulase-negative) isolates.

Objectives: Current information on rates and dynamics of meningococcal carriage is essential for public health policy. This study aimed to determine meningococcal carriage prevalence, its risk factors and duration in the Netherlands, where meningococcal C vaccine coverage is >90%. Several methods to identify serogroups of meningococcal carriage isolates among adolescent and young adults were compared. Methods: Oropharyngeal swabs were collected from 1715 participants 13-23 years of age in 2013-2014; 300 were prospectively followed over 8 months. Cultured isolates were characterized by Ouchterlony, real-time (rt-) PCR or whole-genome sequencing (WGS). Direct swabs were assessed by rt-PCR. Questionnaires on environmental factors and behaviour were also obtained. Results: A meningococcal isolate was identified in 270/1715 (16%) participants by culture. Of MenB isolates identified by whole genome sequencing, 37/72 (51%) were correctly serogrouped by Ouchterlony, 46/51 (90%) by rt-PCR of cultured isolates, and 39/51 (76%) by rt-PCR directly on swabs. A sharp increase in carriage was observed before the age of 15 years. The age-related association disappeared after correction for smoking, level of education, frequent attendance to crowded social venues, kissing in the previous week and alcohol consumption. Three participants carried the same strain identified at three consecutive visits in an 8-month period. In these isolates, progressively acquired mutations were observed. Conclusions: Whole genome sequencing of culture isolates was the most sensitive method for serogroup identification. Based upon results of this study and risk of meningococcal disease, an adolescent meningococcal vaccination might include children before the age of 15 years to confer individual protection and potentially to establish herd protection. (C) 2017 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases

A key aspect of the pathogenesis of the Gram positive bacterium Staphylococcus aureus is its ability to rapidly adapt to the host environment during the course of an infection. To successfully establish infection, the organism deploys a variety of survival and immune evasion strategies, ranging from the acquisition of essential nutrients and expression of adhesins, which promote colonization and survival, to the elaboration of virulence factors such as capsule, which aids host immune evasion. The ability of S. aureus to deploy different virulence factors must be taken into account for S. aureus vaccine design. Here we present a strategy for designing an effective vaccine against S. aureus disease by evaluating vaccine candidate performance in multiple in vivo models targeted to mimic aspects of human disease, and by co-development of functional in vitro immunoassays that measure the neutralization of relevant S. aureus virulence factors.

The streptomycetes, producers of more than half of the 10,000 documented bioactive compounds, have offered over 50 years of interest to industry and academia. Despite this, their taxonomy remains somewhat confused and the definition of species is unresolved due to the variety of morphological, cultural, physiological and biochemical characteristics that are observed at both the inter- and the intraspecies level. This review addresses the current status of streptomycete taxonomy, highlighting the value of a polyphasic approach that utilizes genotypic and phenotypic traits for the delimitation of species within the genus.

Immunofluorescence microscopy is a widely used laboratory method which allows detection and visualization of specific antigens. The method employs the specificity of antibodies to deliver fluorophore to a specific target and then visualize it with a microscope. The power of the technique is that it requires relatively little manipulation and relatively few bacterial cells, enabling the detection of antigen expression where other methods cannot, such as during an actual infection in an animal. Here, we apply the method to follow antigen expression on the surface of MRSA cells over time in in vivo infection models.

Immunofluorescence microscopy is a widely used laboratory method which allows detection and visualization of specific antigens. The method employs the specificity of antibodies to deliver fluorophore to a specific target and then visualize it with a microscope. The power of the technique is that it requires relatively little manipulation and relatively few bacterial cells, enabling the detection of antigen expression where other methods cannot, such as during an actual infection in an animal. Here, we apply the method to follow antigen expression on the surface of MRSA cells over time in in vivo infection models.

SUMMARY Neisseria meningitidis is a Gram-negative microorganism that exists exclusively in humans and can cause devastating invasive disease. Although capsular polysaccharide-based vaccines against serogroups A, C, Y, and W135 are widely available, the pathway to a broadly protective vaccine against serogroup B has been more complex. The last 11 years has seen the discovery and development of the N. meningitidis serogroup B (MnB) outer membrane protein factor H binding protein (fHBP) as a vaccine component. Since the initial discovery of fHBP, a tremendous amount of work has accumulated on the diversity, structure, and regulation of this important protein. fHBP has proved to be a virulence factor for N. meningitidis and a target for functional bactericidal antibodies. fHBP is critical for survival of meningococci in the human host, as it is responsible for the primary interaction with human factor H (fH). Binding of hfH by the meningococcus serves to downregulate the host alternative complement pathway and helps the organism evade host innate immunity. Preclinical studies have shown that an fHBP-based vaccine can elicit serum bactericidal antibodies capable of killing MnB, and the vaccine has shown very encouraging results in human clinical trials. This report reviews our current knowledge of fHBP. In particular, we discuss the recent advances in our understanding of fHBP, its importance to N. meningitidis , and its potential role as a vaccine for preventing MnB disease.

The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10 000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.

Staphylococcus aureus is a leading cause of both healthcare- and community-associated infections globally. S. aureus exhibits diverse clinical presentations, ranging from benign carriage and superficial skin and soft tissue infections to deep wound and organ/space infections, biofilm-related prosthesis infections, life-threatening bacteremia and sepsis. This broad clinical spectrum, together with the high incidence of these disease manifestations and magnitude of the diverse populations at risk, presents a high unmet medical need and a substantial burden to the healthcare system. With the increasing propensity of S. aureus to develop resistance to essentially all classes of antibiotics, alternative strategies, such as prophylactic vaccination to prevent S. aureus infections, are actively being pursued in healthcare settings. Within the last decade, the S. aureus vaccine field has witnessed two major vaccine failures in phase 3 clinical trials designed to prevent S. aureus infections in either patients undergoing cardiothoracic surgery or patients with end-stage renal disease undergoing hemodialysis. This review summarizes the potential underlying reasons why these two approaches may have failed, and proposes avenues that may provide successful vaccine approaches to prevent S. aureus disease in the future.

International audience; BACKGROUND:Recombinant vaccines containing factor H-binding protein (fHBP) have been developed for the purpose of protection from invasive meningococcal serogroup B disease. Neisseria meningitidis fHBP sequences can be divided into 2 genetically and immunologically distinct subfamilies (A and B); thus, cross protection is conferred within but not between subfamilies. A comprehensive understanding of fHBP epidemiology is required to accurately assess the potential vaccine impact when considering different vaccination implementation strategies.METHODS:Systematically collected invasive meningococcal serogroup B isolates from England, Wales, Northern Ireland, the United States, Norway, France and the Czech Republic were previously characterized for fHBP sequence. This study expanded the evaluation with additional meningococcal serogroup B disease isolates from Spain (n = 346) and Germany (n = 205). This expanded set (n = 1841), collected over a 6-year period (2001 to 2006), was evaluated for fHBP sequence and fHBP subfamily relative to patient age.RESULTS:All 1841 isolates contained fhbp. fHBP sequences from Spain and Germany fell within the previously described subfamilies, with 69% of isolates belonging to subfamily B and 31% to subfamily A; prevalent sequence variants were also similar. Stratification of data by age indicated that disease in infants

BACKGROUND: A bivalent, recombinant, factor H-binding protein (rLP2086) vaccine was developed to protect against invasive Neisseria meningitidis serogroup B (MnB) in children and adolescents. METHODS: Healthy toddlers (N = 99) were enrolled to 3 ascending dose-level cohorts (20, 60 or 200 μg). Within each cohort (n = 33), subjects were randomized to receive an initial formulation of the bivalent rLP2086 vaccine at 0, 1 and 6 months or hepatitis A vaccine/placebo control (2:1 ratio). Reactogenicity was assessed by parental reporting of local and systemic reactions using electronic diaries and reports of unsolicited adverse events. Immunogenicity was assessed by serum bactericidal activity assay using human complement and rLP2086-specific IgG binding. RESULTS: The vaccine was considered to be well tolerated. Tenderness was the most frequently reported local reaction. Upper respiratory tract infection was the most commonly reported adverse event and occurred more frequently in the control group. Three cases (200 μg dose) of severe erythema that did not interfere with limb movement were reported. Four toddlers developed fever >40.0°C, 3 in the 200 μg group and 1 in the 60 μg group. Postdose 3, seroconversion (serum bactericidal activity assay using human complement ≥4-fold rise from baseline) was observed in 61.1-88.9% of participants against MnB strains expressing LP2086 variants homologous or nearly homologous to vaccine antigens and 11.1-44.4% against MnB strains expressing heterologous LP2086 variants. Seroconversion was observed in 77.8-100% of participants against additional, exploratory MnB strains expressing vaccine-homologous or heterologous LP2086 variants. CONCLUSIONS: This study shows that the bivalent rLP2086 vaccine is well tolerated and immunogenic in toddlers.

Staphylococci cause diseases ranging from relatively mild superficial skin infections to life-threatening conditions, including sepsis and endocarditis. Increasingly, staphylococci have developed antibiotic resistance, reducing treatment options and highlighting the need for an effective prophylactic vaccine. Preclinical studies have assessed several antigens that, either alone [1, 2] or in combination [3], have the ability to reduce staphylococcal disease in preclinical models. Staphylococcal strains are known for the phenotypic plasticity of their antigenic repertoire, which provides mechanisms for both survival in diverse host niches and for immune evasion. Thus the development of a broadly protective vaccine that can prevent different diseases caused by diverse strains from within the species group poses challenges. S. aureus distinguishes itself from other staphylococci by an impressive array of virulence factors [4] and the production of coagulase. S. epidermidis also causes disease with associated mortality [5, 6]. S. aureus and S. epidermidis share a core genome representing approximatly two thirds of their genes; the proteins encoded by the core genesaverage 70% amino acid sequence identity between the two organisms [4]. We were interested in evaluating proteins with the potential to prevent staphylococcal disease caused by either S. aureus or S. epidermidis. Effective bacterial vaccine components should be conserved and perform important functions during infection. Initial attempts to identify S. aureus vaccine candidates came from assessment of surface antigens from bacteria grown in vitro [7]. Advances in in vivo technology have led to a better understanding of bacterial antigen expression during infection, enhancing our knowledge of host-pathogen relationships. When S. aureus and other pathogenic bacteria invade, they initiate expression of multiple virulence pathways. S. aureus produces capsular polysaccharide to avoid attack from the innate immune system. It also expresses various ligand-binding proteins with roles ranging from immune cloaking [8] to scavenging essential ions [9]. One group of proteins associated with in vivo survival of S. aureus are the manganese transport (Mnt) proteins. The Mnt complex is an ABC transporter composed of an ATP-binding protein (MntA), an integral membrane transporter (MntB), and a manganese binding surface lipoprotein (MntC) [10]. Recently MntC was identified as being expressed on the cell surface of S. aureus in biofilms generated in in vivo models of infection [11]. The orthologous protein in S. epidermidis is staphylococcal iron transport C (SitC) [12], which was detected in convalescent-phase serum from rabbits infected with S. epidermidis and found to be protective in a S. epidermidis murine kidney abscess model [13]. A critical attribute for an effective staphylococcal vaccine antigen is early expression during infection, which provides an opportunity for vaccine-induced antibodies to inactivate bacteria before infection is firmly established. We therefore evaluated S. aureus MntC temporal expression in vivo. S. aureus MntC and S. epidermidis SitC have a high level of sequence identity (72%), and polyclonal antisera have been demonstrated to be cross-reactive [13]. We explored whether S. aureus MntC could produce cross-protective antibodies against both S. aureus and S. epidermidis infection.

Challenges remain in developing effective meningococcal vaccines and vaccination programs in both the industrialized and developing worlds. Vaccination programs should ideally provide broad coverage of disease-causing strains to prevent disease and also promote the herd immunity important for extending protection to the at-risk unvaccinated population. The success of meningococcal serogroup C conjugate vaccination programs during the last 10 years demonstrates the tremendous positive impact on human health that may be attainable; however, significant challenges remain to implement effective use of serogroup A vaccines for the developing world and to develop broadly protective vaccines against serogroup B disease. The effectiveness of vaccination programs, and shifts in disease epidemiology, must be monitored continuously to evaluate protection levels and to identify gaps in disease coverage. As these challenges are met, the possibility for removing the fear of meningococcal disease from all societies is now becoming a reality.

A fully human monoclonal antibody (CS-D7, IgG1) specific for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus was isolated from the Cambridge Antibody Technology (CAT) scFv antibody library. As compared to previously described IsdB specific murine monoclonals, CS-D7 has a unique, non-overlapping binding site on IsdB, and exhibits increased in vivo activity. The antibody recognizes a conformational epitope spanning amino acids 50 to 285 and has a binding affinity of 340 (± 75) pM for IsdB. CS-D7 bound to a wide variety of S. aureus strains, but not to an isdB deletion mutant. The antibody mediated opsonophagocytic (OP) killing in vitro and mediated significant protection in vivo. In a murine lethal sepsis model, the antibody conferred protection from death when dosed prior to challenge, but not when dosed after challenge. Importantly, in a central venous catheter (CVC) model in rats, the antibody reduced bacteremia and prevented colonization of indwelling catheters. Protection was observed when rats were dosed with CS-D7 prior to challenge as well as post challenge. IsdB is currently being investigated for clinical efficacy against S. aureus infection, and the activity of this human IsdB specific antibody supplements the growing body of evidence to support targeting this antigen for vaccine development.

Neisseria meningitidis (Nm) serogroups B, C and Y are the major causes of meningococcal diseases in the United States. NmB accounts for ∼1/3 of the disease but no licensed vaccine is yet available. Two candidate vaccines are being developed specifically to target NmB, but may also provide protection against other serogroups. To assess the potential impact of these vaccines on NmB and other serogroups causing disease in the US, we determined the prevalence, genetic diversity and epidemiological characteristics of three candidate antigen genes in Nm isolates collected through Active Bacterial Core surveillance (ABCs), a population-based active surveillance program. fHbp was detected in all NmB, NmY and NmW135 isolates. Eleven NmC isolates contain fHbp with a single base-pair deletion creating a frame shift in the C-terminal region. Among NmB, 59% were FHbp subfamily/variant B/v1 and 41% A/v2-3. Among NmC and NmY, 39% and 3% were B/v1, respectively. nadA was detected in 39% of NmB, 61% of NmC and 4% of NmY. Among isolates tested, nhbA was present in all NmB and 96% of non-B. For the subset of strains sequenced for NadA and NhbA, pairwise identity was greater than 93% and 78%, respectively. The proportion of FHbp subfamily/variant was different between ABCs site and year, but no linear temporal trend was observed. Although assessment of the vaccine coverage also requires understanding of the antigen expression and the ability to induce bactericidal activity, our finding that all isolates contain one or more antigen genes suggests these candidate vaccines may protect against multiple Nm serogroups.

Factor H binding proteins (fHBP), are bacterial surface proteins currently undergoing human clinical trials as candidate serogroup B Neisseria meningitidis (MnB) vaccines. fHBP protein sequences segregate into two distinct subfamilies, designated A and B. Here, we report the specificity and vaccine potential of mono- or bivalent fHBP-containing vaccines. A bivalent fHBP vaccine composed of a member of each subfamily elicited substantially broader bactericidal activity against MnB strains expressing heterologous fHBP than did either of the monovalent vaccines. Bivalent rabbit immune sera tested in serum bactericidal antibody assays (SBAs) against a diverse panel of MnB clinical isolates killed 87 of the 100 isolates. Bivalent human immune sera killed 36 of 45 MnB isolates tested in SBAs. Factors such as fHBP protein variant, PorA subtype, or MLST were not predictive of whether the MnB strain could be killed by rabbit or human immune sera. Instead, the best predictor for killing in the SBA was the level of in vitro surface expression of fHBP. The bivalent fHBP vaccine candidate induced immune sera that killed MnB isolates representing the major MLST complexes, prevalent PorA subtypes, and fHBP variants that span the breadth of the fHBP phylogenetic tree. Importantly, epidemiologically prevalent fHBP variants from both subfamilies were killed.

Background. Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000–2006. Methods. Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. Results. Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%– 75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. Conclusions. The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST. Invasive disease caused by Neisseria meningitidis is a rapidly progressing, disseminated infection with a case fatality rate of ∼10% [1], and 10%–20% of survivors experience serious permanent sequelae (eg, neurological impairment, digit, limb, or hearing loss). Vaccines for meningococcal serogroups A, C, Y, and W135

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.

Staphylococcal infections associated with catheter and prosthetic implants are difficult to eradicate and often lead to chronic infections. Development of novel antibacterial therapies requires simple, reliable, and relevant models for infection. Using bioluminescent Staphylococcus aureus , we have adapted the existing foreign-body and deep-wound mouse models of staphylococcal infection to allow real-time monitoring of the bacterial colonization of catheters or tissues. This approach also enables kinetic measurements of bacterial growth and clearance in each infected animal. Persistence of infection was observed throughout the course of the study until termination of the experiment at day 16 in a deep-wound model and day 21 in the foreign-body model, providing sufficient time to test the effects of antibacterial compounds. The usefulness of both animal models was assessed by using linezolid as a test compound and comparing bioluminescent measurements to bacterial counts. In the foreign-body model, a three-dose antibiotic regimen (2, 5, and 24 h after infection) resulted in a decrease in both luminescence and bacterial counts recovered from the implant compared to those of the mock-treated infected mice. In addition, linezolid treatment prevented the formation of subcutaneous abscesses, although it did not completely resolve the infection. In the thigh model, the same treatment regimen resulted in complete resolution of the luminescent signal, which correlated with clearance of the bacteria from the thighs.

BackgroundStaphylococcus aureus is a common cause of healthcare-acquired morbidity and mortality and increased healthcare resource utilization. A prophylactic vaccine is being developed that may reduce this disease burden.MethodsVolunteers in good general health aged 50–85 (n=312) and 18–24 (n=96) years were randomized to receive a single intramuscular dose of one of three dose levels of a non-adjuvanted, 3-antigen S. aureus vaccine (SA3Ag) or placebo. SA3Ag antigens included capsular polysaccharides 5 and 8 (CP5 and CP8), each conjugated to cross-reactive material 197 (CRM197), and recombinant clumping factor A (ClfA). Safety, tolerability, and immunogenicity were evaluated.ResultsAt day 29 post-vaccination, robust immune responses were observed in both age cohorts at all three SA3Ag dose levels. In the primary analysis population, the 50- to 85-year age stratum, geometric mean-fold-rises in competitive Luminex® immunoassay antibody titers from baseline ranged from 29.2 to 83.7 (CP5), 14.1 to 31.0 (CP8), and 37.1 to 42.9 (ClfA), all (P