Investigation of cross-opsonic effect leads to the discovery of PPIase-domain containing protein vaccine candidate to prevent infections by Gram-positive ESKAPE pathogens.

BACKGROUND: Enterococcus faecium and Staphylococcus aureus are the Gram-positive pathogens of the ESKAPE group, known to represent a great threat to human health due to their high virulence and multiple resistances to antibiotics. Combined, enterococci and S. aureus account for 26% of healthcare-associated infections and are the most common organisms responsible for blood stream infections. We previously showed that the peptidyl-prolyl cis/trans isomerase (PPIase) PpiC of E. faecium elicits the production of specific, opsonic, and protective antibodies that are effective against several strains of E. faecium and E. faecalis. Due to the ubiquitous characteristics of PPIases and their essential function within Gram-positive cells, we hypothesized a potential cross-reactive effect of anti-PpiC antibodies. RESULTS: Opsonophagocytic assays combined with bioinformatics led to the identification of the foldase protein PrsA as a new potential vaccine antigen in S. aureus. We show that PrsA is a stable dimeric protein able to elicit opsonic antibodies against the S. aureus strain MW2, as well as cross-binding and cross-opsonic in several S. aureus, E. faecium and E. faecalis strains. CONCLUSIONS: Given the multiple antibiotic resistances S. aureus and enterococci present, finding preventive strategies is essential to fight those two nosocomial pathogens. The study shows the potential of PrsA as an antigen to use in vaccine formulation against the two dangerous Gram-positive ESKAPE bacteria. Our findings support the idea that PPIases should be further investigated as vaccine targets in the frame of pan-vaccinomics strategy.

Bacterial Opsonization Changes Adhesion Interactions between Endothelial Cells and Neutrophils in a Model of Experimental Septicemia.

The influence of non-opsonized and opsonized S. aureus 2879M and E. coli 321 strains on the total strength of interaction between the endothelial cell and neutrophil during the docking process was studied using in vitro model of experimental septicemia. We observed a decrease in the force and work of adhesion between receptors of neutrophils and endothelial cells under the influence of non-opsonized strains and further decrease in the affinity of single interactions between cells under the influence of opsonized S. aureus, which was compensated by an increase in the number of contacts, as well as an increase in the force of adhesion under the influence of opsonized E. coli compared to non-opsonized bacteria, which remained below the control level, while adhesion work reaches the control level. Thus, opsonization of S. aureus aggravates the "immunological uncoupling" between neutrophils and endothelial cells, while opsonization of E. coli reduces the pathological effect compared to non-opsonized bacteria.

Mechanisms of anti-GPIbα antibody-induced thrombocytopenia in mice.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by antibody-mediated platelet destruction. Different mechanisms have been suggested to explain accelerated platelet clearance and impaired thrombopoiesis, but the pathophysiology of ITP has yet to be fully delineated. In this study, we tested 2 mouse models of immune-mediated thrombocytopenia using the rat anti-mouse GPIbα monoclonal antibody 5A7, generated in our laboratory. After a single IV administration of high-dose (2 mg/kg) 5A7, opsonized platelets were rapidly cleared from the circulation into the spleen and liver; this was associated with rapid upregulation of thrombopoietin (TPO) messenger RNA. In contrast, subcutaneous administration of low-dose 5A7 (0.08-0.16 mg/kg) every 3 days gradually lowered the platelet count; in this case, opsonized platelets were observed only in the spleen, and TPO levels remained unaltered. Interestingly, in both models, the 5A7 antibody was found on the surface of, as well as internalized to, bone marrow megakaryocytes. Consequently, platelets generated in the chronic phase of repeated subcutaneous 5A7 administration model showed reduced GPIbα membrane expression on their surface. Our findings indicate that evaluation of platelet surface GPIbα relative to platelet size may be a useful marker to support the diagnosis of anti-GPIbα antibody-induced ITP.

Characterization of the Dual Functions of LvCrustinVII from Litopenaeus vannamei as Antimicrobial Peptide and Opsonin.

Crustin are a family of antimicrobial peptides that play an important role in protecting against pathogens infection in the innate immune system of crustaceans. Previously, we identified several novel types of crustins, including type VI and type VII crustins. However, their immune functions were still unclear. In the present study, the immune function of type VII crustin LvCrustinVII were investigated in Litopenaeus vannamei. LvCrustinVII was wildly expressed in all tested tissues, with relatively high expression levels in hepatopancreas, epidermis and lymphoid organ. Upon Vibrio parahaemolyticus infection, LvCrustinVII was significantly upregulated in hepatopancreas. Recombinant LvCrustinVII (rLvCrustinVII) showed strong inhibitory activities against Gram-negative bacteria Vibrio harveyi and V. parahaemolyticus, while weak activities against the Gram-positive bacteria Staphylococcus aureus. Binding assay showed that rLvCrustinVII could bind strongly to V. harveyi and V. parahaemolyticus, as well as the cell wall components Glu, LPS and PGN. In the presence of Ca2+, rLvCrustinVII could agglutinate V. parahaemolyticus and enhance hemocyte phagocytosis. The present data partially illustrate the immune function of LvCrustinVII, which enrich our understanding on the functional mechanisms of crustins and provide useful information for application of this kind of antimicrobial peptides.

Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation.

Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-alpha1beta2, and the cells had low lysosomal enzyme expression and retained opsonized antigens on their surface. Intravital imaging revealed immune complexes moving along macrophage processes into the follicle. Moreover, noncognate B cells relayed antigen opsonized by newly produced antibodies from the subcapsular region to the germinal center, and affinity maturation was impaired when this transport process was disrupted. Thus, we characterize SCS macrophages as specialized antigen-presenting cells functioning at the apex of an antigen transport chain that promotes humoral immunity.

Complement-Opsonized Nano-Carriers Are Bound by Dendritic Cells (DC) via Complement Receptor (CR)3, and by B Cell Subpopulations via CR-1/2, and Affect the Activation of DC and B-1 Cells.

The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.

Antibody Targets on the Surface of Plasmodium falciparum-Infected Erythrocytes That Are Associated With Immunity to Severe Malaria in Young Children.

BACKGROUND: Sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the microvasculature contributes to pathogenesis of severe malaria in children. This mechanism is mediated by antigens expressed on the IE surface. However, knowledge of specific targets and functions of antibodies to IE surface antigens that protect against severe malaria is limited. METHODS: Antibodies to IE surface antigens were examined in a case-control study of young children in Papua New Guinea presenting with severe or uncomplicated malaria (n = 448), using isolates with a virulent phenotype associated with severe malaria, and functional opsonic phagocytosis assays. We used genetically modified isolates and recombinant P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains to quantify PfEMP1 as a target of antibodies associated with disease severity. RESULTS: Antibodies to the IE surface and recombinant PfEMP1 domains were significantly higher in uncomplicated vs severe malaria and were boosted following infection. The use of genetically modified P. falciparum revealed that PfEMP1 was a major target of antibodies and that PfEMP1-specific antibodies were associated with reduced odds of severe malaria. Furthermore, antibodies promoting the opsonic phagocytosis of IEs by monocytes were lower in those with severe malaria. CONCLUSIONS: Findings suggest that PfEMP1 is a dominant target of antibodies associated with reduced risk of severe malaria, and function in part by promoting opsonic phagocytosis.

Development of Opsonic Mouse Monoclonal Antibodies against Multidrug-Resistant Enterococci.

Multidrug-resistant enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (MAbs) targeting bacterial antigens would be a valuable treatment option in this setting. Here, we describe the development of two MAbs through hybridoma technology that target antigens from the most clinically relevant enterococcal species. Diheteroglycan (DHG), a well-characterized capsular polysaccharide of Enterococcus faecalis, and the secreted antigen A (SagA), an immunogenic protein from Enterococcus faecium, are both immunogens that have been proven to raise opsonic and cross-reactive antibodies against enterococcal strains. For this purpose, a conjugated form of the native DHG with SagA was used to raise the antibodies in mice, while enzyme-linked immunosorbent assay and opsonophagocytic assay were combined in the selection process of hybridoma cells producing immunoreactive and opsonic antibodies targeting the selected antigens. From this process, two highly specific IgG1(κ) MAbs were obtained, one against the polysaccharide (DHG.01) and one against the protein (SagA.01). Both MAbs exhibited good opsonic killing against the target bacterial strains: DHG.01 showed 90% killing against E. faecalis type 2, and SagA.01 showed 40% killing against E. faecium 11231/6. In addition, both MAbs showed cross-reactivity toward other E. faecalis and E. faecium strains. The sequences from the variable regions of the heavy and light chains were reconstructed in expression vectors, and the activity of the MAbs upon expression in eukaryotic cells was confirmed with the same immunological assays. In summary, we identified two opsonic MAbs against enterococci which could be used for therapeutic or prophylactic approaches against enterococcal infections.

Multi-component meningococcal serogroup B (MenB)-4C vaccine induces effective opsonophagocytic killing in children with a complement deficiency.

Vaccination against meningococcal serogroup B is recommended for patients with a complement deficiency; however, although immunogenicity in this patient group has been shown, efficacy has not yet been established. In this study, we collected serum from children with a complement deficiency in the alternative pathway or in late terminal pathway before and after vaccination with multi-component meningococcal serogroup B (MenB)-4C. MenB-4C is a multi-component, protein-based vaccine against MenB consisting of factor H-binding protein, Neisserial heparin-binding protein, Neisserial adhesion A and outer membrane vesicles containing Porin A. We assessed the vaccine immunogenicity and vaccine-mediated protection by a whole cell enzyme-linked immunosorbent assay with Neisseria meningitidis serogroup B strains H44/76, 5/99 and NZ98/254, which shows that vaccination induced antibody titers against meningococcus. We show that the classical serum bactericidal activity assay with exogenous serum indicates the presence of vaccine-induced antibodies and capacity to activate complement-mediated pathogen lysis. However, in children with a late terminal pathway deficiency, no complement-mediated pathogen lysis was observed when autologous serum was applied in the serum bactericidal activity assay, demonstrating a lack of serum bactericidal activity in children with complement deficiencies. However, MenB-4C vaccination still induced effective complement-dependent opsonophagocytic killing against N. meningitidis serogroup B in reconstituted whole blood with autologous serum from children with an alternative pathway or late terminal pathway deficiency. These findings support the recommendation to vaccinate all complement-deficient children against MenB.

Serum IgA Immune Complexes Promote Proinflammatory Cytokine Production by Human Macrophages, Monocytes, and Kupffer Cells through FcαRI-TLR Cross-Talk.

IgA is predominantly recognized to play an important role in host defense at mucosal sites, where it prevents invasion of pathogens by neutralization. Although it has recently become clear that IgA also mediates other immunological processes, little remains known about the potential of IgA to actively contribute to induction of inflammation, particularly in nonmucosal organs and tissues. In this article, we provide evidence that immune complex formation of serum IgA plays an important role in orchestration of inflammation in response to pathogens at various nonmucosal sites by eliciting proinflammatory cytokines by human macrophages, monocytes, and Kupffer cells. We show that opsonization of bacteria with serum IgA induced cross-talk between FcαRI and different TLRs, leading to cell type-specific amplification of proinflammatory cytokines, such as TNF-α, IL-1ß, IL-6, and IL-23. Furthermore, we demonstrate that the increased protein production of cytokines was regulated at the level of gene transcription, which was dependent on activation of kinases Syk and PI3K. Taken together, these data demonstrate that the immunological function of IgA is substantially more extensive than previously considered and suggest that serum IgA-induced inflammation plays an important role in orchestrating host defense by different cell types in nonmucosal tissues, including the liver, skin, and peripheral blood.

Novel Immunoprotective Proteins of Streptococcus pneumoniae Identified by Opsonophagocytosis Killing Screen.

The success of polysaccharide conjugate vaccines represents a major advance in the prevention of pneumococcal disease, but the power of these vaccines is limited by partial spectrum of coverage and high cost. Vaccines using immunoprotective proteins are a promising alternative type of pneumococcal vaccines. In this study, we constructed a library of antisera against conserved pneumococcal proteins predicted to be associated with cell surface or virulence using a combination of bioinformatic prediction and immunization of rabbits with recombinant proteins. Screening of the library by an opsonophagocytosis killing (OPK) assay identified the OPK-positive antisera, which represented 15 (OPK-positive) proteins. Further tests showed that virtually all of these OPK-positive antisera conferred passive protection against lethal infection of virulent pneumococci. More importantly, immunization with recombinant forms of three OPK-positive proteins (SP148, PBP2b, and ScpB), alone or in combination, conferred significant protection against lethal challenge of pneumococcal strains representing capsular serotypes 3, 4, and 6A in a mouse sepsis model. To our best knowledge, this work represents the first example in which novel vaccine candidates are successfully identified by the OPK screening. Our data have also provided further confirmation that the OPK activity may serve as a reliable in vitro surrogate for evaluating vaccine efficacy of pneumococcal proteins.

Plasmodium falciparum MSP3 Exists in a Complex on the Merozoite Surface and Generates Antibody Response during Natural Infection.

Plasmodium falciparum merozoite surface protein 3 (MSP3) is an abundantly expressed secreted merozoite surface protein and a leading malaria vaccine candidate antigen. However, it is unclear how MSP3 is retained on the surface of merozoites without a glycosylphosphatidylinositol (GPI) anchor or a transmembrane domain. In the present study, we identified an MSP3-associated network on the Plasmodium merozoite surface by immunoprecipitation of Plasmodium merozoite lysate using antibody to the N terminus of MSP3 (anti-MSP3N) followed by mass spectrometry analysis. The results suggested the association of MSP3 with other merozoite surface proteins: MSP1, MSP6, MSP7, RAP2, and SERA5. Protein-protein interaction studies by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis showed that MSP3 complex consists of MSP1, MSP6, and MSP7 proteins. Immunological characterization of MSP3 revealed that MSP3N is strongly recognized by hyperimmune serum from African and Asian populations. Furthermore, we demonstrate that human antibodies, affinity purified against recombinant MSP3N (rMSP3N), promote opsonic phagocytosis of merozoites in cooperation with monocytes. At nonphysiological concentrations, anti-MSP3N antibodies inhibited the growth of P. falciparum in vitro Together, the data suggest that MSP3 and especially its N-terminal region containing known B/T cell epitopes are targets of naturally acquired immunity against malaria and also comprise an important candidate for a multisubunit malaria vaccine.

Development of flow cytometric opsonophagocytosis and antibody-mediated complement deposition assays for non-typeable Haemophilus influenzae.

BACKGROUND: Haemophilus influenzae is found in the nasopharynx of 80% of the human population. While colonisation with non-typeable Haemophilus influenzae (NTHi) is usually asymptomatic, it is capable of causing acute and chronic otitis media (OM) in infants, invasive disease in susceptible groups and is the leading cause of exacerbations of patients with chronic obstructive pulmonary disease (COPD). Current methods for assessing functional antibody immunity to NTHi are limited and labour intensive. Flow cytometric assays could provide an attractive alternative to evaluate immune responses to candidate vaccines in clinical trials. RESULTS: We have developed a duplexed flow-cytometric uptake and oxidative burst opsonophagocytosis assay (fOPA). We have also developed a duplexed antibody-mediated complement C3b/iC3b and C5b-9 deposition assay (CDA). Antibody-mediated C3b/iC3b deposition correlated with opsonophagocytic uptake (r = 0.65) and with opsonophagocytic oxidative burst (r = 0.69). Both fOPA and CDA were reproducible, with the majority of samples giving a coefficient of variation (CV) of < 20% and overall assay CVs of 14% and 16% respectively. CONCLUSIONS: The high-throughput flow cytometric assays developed here were successfully optimised for use with NTHi. Assays proved to be sensitive and highly reproducible for the measurement of bacterial uptake and oxidative burst opsonophagocytosis and antibody-mediated deposition of C3b/iC3b and C5b-9. These assays are useful tools for use in large scale epidemiological studies and to assist in the assessment of functional antibody induced by NTHi candidate vaccines.

Beyond the Matrix: The Many Non-ECM Ligands for Integrins.

The traditional view of integrins portrays these highly conserved cell surface receptors as mediators of cellular attachment to the extracellular matrix (ECM), and to a lesser degree, as coordinators of leukocyte adhesion to the endothelium. These canonical activities are indispensable; however, there is also a wide variety of integrin functions mediated by non-ECM ligands that transcend the traditional roles of integrins. Some of these unorthodox roles involve cell-cell interactions and are engaged to support immune functions such as leukocyte transmigration, recognition of opsonization factors, and stimulation of neutrophil extracellular traps. Other cell-cell interactions mediated by integrins include hematopoietic stem cell and tumor cell homing to target tissues. Integrins also serve as cell-surface receptors for various growth factors, hormones, and small molecules. Interestingly, integrins have also been exploited by a wide variety of organisms including viruses and bacteria to support infectious activities such as cellular adhesion and/or cellular internalization. Additionally, the disruption of integrin function through the use of soluble integrin ligands is a common strategy adopted by several parasites in order to inhibit blood clotting during hematophagy, or by venomous snakes to kill prey. In this review, we strive to go beyond the matrix and summarize non-ECM ligands that interact with integrins in order to highlight these non-traditional functions of integrins.

Complement Opsonization Promotes Herpes Simplex Virus 2 Infection of Human Dendritic Cells.

UNLABELLED: Herpes simplex virus 2 (HSV-2) is one of the most common sexually transmitted infections globally, with a very high prevalence in many countries. During HSV-2 infection, viral particles become coated with complement proteins and antibodies, both present in genital fluids, which could influence the activation of immune responses. In genital mucosa, the primary target cells for HSV-2 infection are epithelial cells, but resident immune cells, such as dendritic cells (DCs), are also infected. DCs are the activators of the ensuing immune responses directed against HSV-2, and the aim of this study was to examine the effects opsonization of HSV-2, either with complement alone or with complement and antibodies, had on the infection of immature DCs and their ability to mount inflammatory and antiviral responses. Complement opsonization of HSV-2 enhanced both the direct infection of immature DCs and their production of new infectious viral particles. The enhanced infection required activation of the complement cascade and functional complement receptor 3. Furthermore, HSV-2 infection of DCs required endocytosis of viral particles and their delivery into an acid endosomal compartment. The presence of complement in combination with HSV-1- or HSV-2-specific antibodies more or less abolished HSV-2 infection of DCs. Our results clearly demonstrate the importance of studying HSV-2 infection under conditions that ensue in vivo, i.e., conditions under which the virions are covered in complement fragments and complement fragments and antibodies, as these shape the infection and the subsequent immune response and need to be further elucidated. IMPORTANCE: During HSV-2 infection, viral particles should become coated with complement proteins and antibodies, both present in genital fluids, which could influence the activation of the immune responses. The dendritic cells are activators of the immune responses directed against HSV-2, and the aim of this study was to examine the effects of complement alone or complement and antibodies on HSV-2 infection of dendritic cells and their ability to mount inflammatory and antiviral responses. Our results demonstrate that the presence of antibodies and complement in the genital environment can influence HSV-2 infection under in vitro conditions that reflect the in vivo situation. We believe that our findings are highly relevant for the understanding of HSV-2 pathogenesis.

Structural Basis for the Function of Complement Component C4 within the Classical and Lectin Pathways of Complement.

Complement component C4 is a central protein in the classical and lectin pathways within the complement system. During activation of complement, its major fragment C4b becomes covalently attached to the surface of pathogens and altered self-tissue, where it acts as an opsonin marking the surface for removal. Moreover, C4b provides a platform for assembly of the proteolytically active convertases that mediate downstream complement activation by cleavage of C3 and C5. In this article, we present the crystal and solution structures of the 195-kDa C4b. Our results provide the molecular details of the rearrangement accompanying C4 cleavage and suggest intramolecular flexibility of C4b. The conformations of C4b and its paralogue C3b are shown to be remarkably conserved, suggesting that the convertases from the classical and alternative pathways are likely to share their overall architecture and mode of substrate recognition. We propose an overall molecular model for the classical pathway C5 convertase in complex with C5, suggesting that C3b increases the affinity for the substrate by inducing conformational changes in C4b rather than a direct interaction with C5. C4b-specific features revealed by our structural studies are probably involved in the assembly of the classical pathway C3/C5 convertases and C4b binding to regulators.

Quantitative proteome analysis of temporally resolved phagosomes following uptake via key phagocytic receptors.

Macrophages operate at the forefront of innate immunity and their discrimination of foreign versus "self" particles is critical for a number of responses including efficient pathogen killing, antigen presentation, and cytokine induction. In order to efficiently destroy the particles and detect potential threats, macrophages express an array of receptors to sense and phagocytose prey particles. In this study, we accurately quantified a proteomic time-course of isolated phagosomes from murine bone marrow-derived macrophages induced by particles conjugated to seven different ligands representing pathogen-associated molecular patterns, immune opsonins or apoptotic cell markers. We identified a clear functional differentiation over the three timepoints and detected subtle differences between certain ligand-phagosomes, indicating that triggering of receptors through a single ligand type has mild, but distinct, effects on phagosome proteome and function. Moreover, our data shows that uptake of phosphatidylserine-coated beads induces an active repression of NF-κB immune responses upon Toll-like receptor (TLR)-activation by recruitment of anti-inflammatory regulators to the phagosome. This data shows for the first time a systematic time-course analysis of bone marrow-derived macrophages phagosomes and how phagosome fate is regulated by the receptors triggered for phagocytosis.

Opsonophagocytic Antibodies to Serotype Ia, Ib, and III Group B Streptococcus among Korean Infants and in Intravenous Immunoglobulin Products.

Group B streptococcus (GBS) infection is a leading cause of sepsis and meningitis among infants, and is associated with high rates of morbidity and mortality in many countries. Protection against GBS typically involves antibody-mediated opsonization by phagocytes and complement components. The present study evaluated serotype-specific functional antibodies to GBS among Korean infants and in intravenous immunoglobulin (IVIG) products. An opsonophagocytic killing assay (OPA) was used to calculate the opsonization indices (OIs) of functional antibodies to serotypes Ia, Ib, and III in 19 IVIG products from 5 international manufacturers and among 98 Korean infants (age: 0-11 months). The GBS Ia, Ib, and III serotypes were selected because they are included in a trivalent GBS vaccine formulation that is being developed. The OI values for the IVIG products were 635-5,706 (serotype Ia), 488-1,421 (serotype Ib), and 962-3,315 (serotype III), and none of the IVIG lots exhibited undetectable OI values (< 4). The geometric mean OI values were similar for all 3 serotypes when we compared the Korean manufacturers. The seropositive rate among infants was significantly lower for serotype Ia (18.4%), compared to serotype Ib and serotype III (both, 38.8%). Infant age of ≥ 3 months was positively correlated with the seropositive rates for each serotype. Therefore, only a limited proportion of infants exhibited protective immunity against serotype Ia, Ib, and III GBS infections. IVIG products that exhibit high antibody titers may be a useful therapeutic or preventive measure for infants. Further studies are needed to evaluate additional serotypes and age groups.

Association of Human Antibodies to Arabinomannan With Enhanced Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction.

BACKGROUND: The relevance of antibodies (Abs) in the defense against Mycobacterium tuberculosis infection remains uncertain. We investigated the role of Abs to the mycobacterial capsular polysaccharide arabinomannan (AM) and its oligosaccharide (OS) fragments in humans. METHODS: Sera obtained from 29 healthy adults before and after primary or secondary bacillus Calmette-Guerin (BCG) vaccination were assessed for Ab responses to AM via enzyme-linked immunosorbent assays, and to AM OS epitopes via novel glycan microarrays. Effects of prevaccination and postvaccination sera on BCG phagocytosis and intracellular survival were assessed in human macrophages. RESULTS: Immunoglobulin G (IgG) responses to AM increased significantly 4-8 weeks after vaccination (P < .01), and sera were able to opsonize BCG and M. tuberculosis grown in both the absence and the presence of detergent. Phagocytosis and intracellular growth inhibition were significantly enhanced when BCG was opsonized with postvaccination sera (P < .01), and these enhancements correlated significantly with IgG titers to AM (P < .05), particularly with reactivity to 3 AM OS epitopes (P < .05). Furthermore, increased phagolysosomal fusion was observed with postvaccination sera. CONCLUSIONS: Our results provide further evidence for a role of Ab-mediated immunity to tuberculosis and suggest that IgG to AM, especially to some of its OS epitopes, could contribute to the defense against mycobacterial infection in humans.

A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium.

The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM197 carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection.