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.

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.

Use of Flow Cytometry to Evaluate Phagocytosis of Staphylococcus aureus by Human Neutrophils.

Neutrophils play a key role in the human immune response to Staphylococcus aureus infections. These professional phagocytes rapidly migrate to the site of infection to engulf bacteria and destroy them via specialized intracellular killing mechanisms. Here we describe a robust and relatively high-throughput flow cytometry assay to quantify phagocytosis of S. aureus by human neutrophils. We show that effective phagocytic uptake of S. aureus is greatly enhanced by opsonization, i.e. the tagging of microbial surfaces with plasma-derived host proteins like antibodies and complement. Our rapid assay to monitor phagocytosis can be used to study neutrophil deficiencies and bacterial evasion, but also provides a powerful tool to assess the opsonic capacity of antibodies, either in the context of natural immune responses or immune therapies.

Opsonins and Dysopsonins of Nanoparticles: Facts, Concepts, and Methodological Guidelines.

Understanding the effects mediated by a set of nanoparticle (NP)-bound host biomolecules, often indicated with the umbrella term of NP corona, is essential in nanomedicine, nanopharmacology, and nanotoxicology. Among the NP-adsorbed proteome, some factors mediate cell binding, endocytosis, and clearing by macrophages and other phagocytes (opsonins), while some others display few affinities for the cell surface (dysopsonins). The functional mapping of opsonins and dysopsonins is instrumental to design long-circulating and nanotoxicologically safe next-generation nanotheranostics. In this review, we critically analyze functional data identifying specific proteins with opsonin or dysopsonin properties. Special attention is dedicated to the following: (1) the simplicity or complexity of the NP proteome and its modulation, (2) the role of specific host proteins in mediating the stealth properties of uncoated or polymer-coated NPs, and (3) the ability of the innate immune system, and, in particular, of the complement proteins, to mediate NP clearance by phagocytes. Emerging species-specific peculiarities, differentiating humans from preclinical animal models (the murine especially), are highlighted throughout this overview. The operative definition of opsonin and dysopsonin and the measurement schemes to assess their in vitro efficacy is critically re-examined. This provides a shared and unbiased approach useful for NP opsonin and dysopsonin systematic identification.

Complement opsonization of HIV affects primary infection of human colorectal mucosa and subsequent activation of T cells.

HIV transmission via genital and colorectal mucosa are the most common routes of dissemination. Here, we explored the effects of free and complement-opsonized HIV on colorectal tissue. Initially, there was higher antiviral responses in the free HIV compared to complement-opsonized virus. The mucosal transcriptional response at 24 hr revealed the involvement of activated T cells, which was mirrored in cellular responses observed at 96 hr in isolated mucosal T cells. Further, HIV exposure led to skewing of T cell phenotypes predominantly to inflammatory CD4+ T cells, that is Th17 and Th1Th17 subsets. Of note, HIV exposure created an environment that altered the CD8+ T cell phenotype, for example expression of regulatory factors, especially when the virions were opsonized with complement factors. Our findings suggest that HIV-opsonization alters the activation and signaling pathways in the colorectal mucosa, which promotes viral establishment by creating an environment that stimulates mucosal T cell activation and inflammatory Th cells.

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens.

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to opsono-phagocytic killing assays. An advantage of the opsono-adherence assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.

Flow Cytometry-Based Assays to Quantify Complement Deposition and Neutrophil Uptake of Group A Streptococcus.

Quantification of complement deposition and subsequent neutrophil-mediated uptake provides an important way to assess the role of different bacterial factors in evasion of the host innate immune response. Here, we describe flow cytometry-based methods to allow quantification of deposition of the complement opsonin C3 on the bacterial surface and subsequent uptake by primary human neutrophils. The assays outlined below provide key methods to determine whether specific bacterial factors are involved in the evasion of complement-mediated immunity, using widely accessible reagents and equipment.

An Opsonophagocytic Killing Assay for the Evaluation of Group A Streptococcus Vaccine Antisera.

Group A Streptococcus (GAS) is a major cause of global mortality, yet there are no licensed GAS vaccines. Vaccine progress has been hampered, in part, by a lack of standardized assays able to quantify antibody function in test antisera. The most widely used assay was developed over 50 years ago by Rebecca Lancefield and relies on human whole blood as a source of complement and neutrophils. Recently, an opsonophagocytic killing (OPK) assay has been developed for GAS by adapting the OPK methods utilized in Streptococcus pneumoniae vaccine testing. This assay uses dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60 cells) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in the Lancefield assays. This protocol outlines methods for performing a GAS OPK assay including titering test sera to generate an opsonic index. This in vitro assay could aid in selecting vaccine candidates by demonstrating whether candidate-induced antibodies lead to complement deposition and opsonophagocytic killing.

Vaccine-Induced Th1-Type Response Protects against Invasive Group A Streptococcus Infection in the Absence of Opsonizing Antibodies.

Recent global advocacy efforts have highlighted the importance of development of a vaccine against group A Streptococcus (GAS). Combo5 is a non-M protein-based vaccine that provides protection against GAS skin infection in mice and reduces the severity of pharyngitis in nonhuman primates. However, Combo5 with the addition of aluminum hydroxide (alum) as an adjuvant failed to protect against invasive GAS infection of mice. Here, we show that formulation of Combo5 with adjuvants containing saponin QS21 significantly improves protective efficacy, even though all 7 adjuvants tested generated high antigen-specific IgG antibody titers, including alum. Detailed characterization of Combo5 formulated with SMQ adjuvant, a squalene-in-water emulsion containing a TLR4 agonist and QS21, showed significant differences from the results obtained with alum in IgG subclasses generated following immunization, with an absence of GAS opsonizing antibodies. SMQ, but not alum, generated strong interleukin-6 (IL-6), gamma interferon (IFN-γ), and tumor necrosis alpha (TNF-α) responses. This work highlights the importance of adjuvant selection for non-M protein-based GAS vaccines to optimize immune responses and protective efficacy.IMPORTANCE Availability of a group A Streptococcus vaccine remains an unmet public health need. Here, we tested different adjuvant formulations to improve the protective efficacy of non-M protein vaccine Combo5 in an invasive disease model. We show that novel adjuvants can dramatically shape the type of immune response developed following immunization with Combo5 and significantly improve protection. In addition, protection afforded by Combo5 is not mediated by opsonizing antibodies, believed to be the main correlate of protection against GAS infections. Overall, this report highlights the importance of adjuvant selection in raising protective immune responses against GAS invasive infection. Adjuvants that can provide a more balanced Th1/Th2-type response may be required to optimize protection of GAS vaccines, particularly those based on non-M protein antigens.

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.

Calreticulin and Galectin-3 Opsonise Bacteria for Phagocytosis by Microglia.

Opsonins are soluble, extracellular proteins, released by activated immune cells, and when bound to a target cell, can induce phagocytes to phagocytose the target cell. There are three known classes of opsonin: antibodies, complement factors and secreted pattern recognition receptors, but these have limited access to the brain. We identify here two novel opsonins of bacteria, calreticulin, and galectin-3 (both lectins that can bind lipopolysaccharide), which were released by microglia (brain-resident macrophages) when activated by bacterial lipopolysaccharide. Calreticulin and galectin-3 both bound to Escherichia coli, and when bound increased phagocytosis of these bacteria by microglia. Furthermore, lipopolysaccharide-induced microglial phagocytosis of E. coli bacteria was partially inhibited by: sugars, an anti-calreticulin antibody, a blocker of the calreticulin phagocytic receptor LRP1, a blocker of the galectin-3 phagocytic receptor MerTK, or simply removing factors released from the microglia, indicating this phagocytosis is dependent on extracellular calreticulin and galectin-3. Thus, calreticulin and galectin-3 are opsonins, released by activated microglia to promote clearance of bacteria. This innate immune response of microglia may help clear bacterial infections of the brain.

Synthetic and biological identities of polymeric nanoparticles influencing the cellular delivery: An immunological link.

Enhanced understanding of bio-nano interaction requires recognition of hidden factors such as protein corona, a layer of adsorbed protein around nano-systems. This study compares the biological identity and fingerprint profile of adsorbed proteins on PLGA-based nanoparticles through nano-liquid chromatography-tandem mass spectrometry. The total proteins identified in the corona of nanoparticles (NPs) with different in size, charge and compositions were classified based on molecular mass, isoelectric point and protein function. A higher abundance of complement proteins was observed in modified NPs with an increased size, while NPs with a positive surface charge exhibited the minimum adsorption for immunoglobulin proteins. A correlation of dysopsonin/opsonin ratio was found with cellular uptake of NPs exposed to two positive and negative Fc receptor cell lines. Although the higher abundance of dysopsonins such as apolipoproteins may cover the active sites of opsonins causing a lower uptake, the correlation of adsorbed dysopsonin/opsonin proteins on the NPs surface has an opposite trend with the intensity of cell uptake. Despite the reduced uptake of corona-coated NPs in comparison with pristine NPs, the dysopsonin/opsonin ratio controlled by the physicochemistry properties of NPs could potentially be used to tune up the cellular delivery of polymeric NPs.

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.

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.

A quantitative Streptococcus pyogenes-human protein-protein interaction map reveals localization of opsonizing antibodies.

A fundamental challenge in medical microbiology is to characterize the dynamic protein-protein interaction networks formed at the host-pathogen interface. Here, we generate a quantitative interaction map between the significant human pathogen, Streptococcus pyogenes, and proteins from human saliva and plasma obtained via complementary affinity-purification and bacterial-surface centered enrichment strategies and quantitative mass spectrometry. Perturbation of the network using immunoglobulin protease cleavage, mixtures of different concentrations of saliva and plasma, and different S. pyogenes serotypes and their isogenic mutants, reveals how changing microenvironments alter the interconnectivity of the interaction map. The importance of host immunoglobulins for the interaction with human complement proteins is demonstrated and potential protective epitopes of importance for phagocytosis of S. pyogenes cells are localized. The interaction map confirms several previously described protein-protein interactions; however, it also reveals a multitude of additional interactions, with possible implications for host-pathogen interactions involving other bacterial species.

Immunization with merozoite surface protein 2 fused to a Plasmodium-specific carrier protein elicits strain-specific and strain-transcending, opsonizing antibody.

Vaccine trials and cohort studies in Plasmodium falciparum endemic areas indicate that naturally-acquired and vaccine-induced antibodies to merozoite surface protein 2 (MSP2) are associated with resistance to malaria. These data indicate that PfMSP2 has significant potential as a component of a multi-antigen malaria vaccine. To overcome challenges encountered with subunit malaria vaccines, we established that the use of highly immunogenic rPfMSP8 as a carrier protein for leading vaccine candidates rPfMSP119 and rPfs25 facilitated antigen production, minimized antigenic competition and enhanced induction of functional antibodies. We applied this strategy to optimize a rPfMSP2 (3D7)-based subunit vaccine by producing unfused rPfMSP2 or chimeric rPfMSP2/8 in Escherichia coli. rPfMSP2 formed fibrils, which induced splenocyte proliferation in an antigen receptor-independent, TLR2-dependent manner. However, fusion to rPfMSP8 prevented rPfMSP2 amyloid-like fibril formation. Immunization of rabbits elicited high-titer anti-PfMSP2 antibodies that recognized rPfMSP2 of the 3D7 and FC27 alleles, as well as native PfMSP2. Competition assays revealed a difference in the specificity of antibodies induced by the two rPfMSP2-based vaccines, with evidence of epitope masking by rPfMSP2-associated fibrils. Rabbit anti-PfMSP2/8 was superior to rPfMSP2-elicited antibody at opsonizing P. falciparum merozoites for phagocytosis. These data establish rPfMSP8 as an effective carrier for a PfMSP2-based subunit malaria vaccine.

Low-density lipoprotein as an opsonin promoting the phagocytosis of Pseudomonas aeruginosa by U937 cells.

Low-density lipoprotein (LDL) was recently reported to be an opsonin, enhancing the phagocytosis of group A Streptococcus (GAS) by human monocytic leukemia U937 cells due to the binding of LDL to some GAS strains. We postulated that LDL might also promote the opsonophagocytosis of Pseudomonas aeruginosa by U937 cells since this bacterium interacts with LDL. In this study, P. aeruginosa (CMCC10104), U937 cells, and human LDL were used in phagocytosis assays to test our hypothesis. Escherichia coli strain BL21, which does not interact with LDL, was used as a negative control. Colony counting and fluorescence microscopy were used to determine the bacterial quantity in the opsonophagocytosis assays. After incubation of U937 cells and P. aeruginosa with LDL (100 µg/ml) for 15 and 30 min, phagocytosis was observed to be increased by 22.71% and 32.90%, respectively, compared to that seen in the LDL-free group. However, LDL did not increase the phagocytosis of E. coli by U937 cells. In addition, we identified CD36 as a major opsonin receptor on U937 cells, since an anti-CD36 monoclonal antibody, but not an anti-CD4 monoclonal antibody, almost completely abolished the opsonophagocytosis of P. aeruginosa by U937 cells.

A C1qDC (CgC1qDC-6) with a collagen-like domain mediates hemocyte phagocytosis and migration in oysters.

Most of the bivalve C1q domain containing proteins (C1qDCs) are either only composed of the globular head domain, or contain an N-terminal coiled-coil domain, presumed to cover a role in oligomerization. On the other hand, collagen regions, widespread in vertebrate C1qDCs, are very uncommon in bivalves. In the present study, a C1qDC with a collagen-like domain (designated CgC1qDC-6) was identified from the Pacific oyster Crassostrea gigas and its possible involvement in immune responses was also characterized. The coding sequence of CgC1qDC-6 was of 756 bp, encoding a peptide of 251 amino acids with an N-terminal signal peptide, a central collagen-like domain, and a C-terminal ghC1q domain. CgC1qDC-6 was clustered with the C1qDCs from several mollusks in the phylogenetic tree. CgC1qDC-6 was detected at both mRNA and protein levels in all tested tissues including hepatopancreas, gonad, gill, mantle, adductor muscle, and hemocytes. The recombinant CgC1qDC-6 protein (rCgC1qDC-6) exhibited binding activity to various pathogen-associated molecular patterns (PAMPs) including LPS, PGN, mannose and Poly I:C, and microorganisms including Gram-negative bacteria (Escherichia coli and Vibrio splendidus), Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus), and fungus (Pichia pastoris). The phagocytic rates of oyster hemocytes towards V. splendidus pre-incubation with rCgC1qDC-6 were significantly enhanced (p < 0.05). In the chemotaxis assay, rCgC1qDC-6 could mediate the migration of oyster hemocytes in a dose-dependent manner, which exhibited a positive chemotactic effect at low concentration (<10 nM). These results collectively indicated that CgC1qDC-6 could serve as a pattern recognition receptor and mediate the hemocyte phagocytosis and migration to eliminate the invading pathogens.

Hemolymph C1qDC promotes the phagocytosis of oyster Crassostrea gigas hemocytes by interacting with the membrane receptor ß-integrin.

Phagocytosis constitutes a conserved cellular process for multicellular animals to ingest or engulf other cells or particles, which is facilitated by the use of opsonins to bind foreign particles and interact with cell surface receptors. The invertebrate secreted C1q domain-containing proteins (C1qDCs) have been reported to exhibit opsonic activity, while the detailed mechanisms of opsonization still remain unclear. In the present study, a C1qDC (designated as CgC1qDC-5) with opsonic activity was identified from the hemolymph of oyster Crassostrea gigas. CgC1qDC-5 exhibited the ability to bind pathogen-associated molecular patterns (PAMPs) of lipopolysaccharides (LPS) and Lipid A. It could also bind and agglutinate Gram-negative bacteria Escherichia coli, Vibrio splendidus and Vibrio anguillarum, whereas the agglutinating activity could be inhibited by LPS. In addition, CgC1qDC-5 could enhance the phagocytosis of hemocytes toward E. coli, V. splendidus, and V. anguillarum. GST pull-down and surface plasmon resonance assays in vitro revealed that CgC1qDC-5 could interact with ß-integrin (CgIntegrin). In vivo, CgC1qDC-5 was observed to bind hemocytes and co-localized with CgIntegrin on the cell membrane of hemocytes. Antibody-mediated blockage of CgIntegrin hindered the CgC1qDC-5-enhanced hemocytic phagocytosis. CgIntegrin also exhibited the ability to bind the Gram-negative bacteria E. coli, V. splendidus, V. anguillarum and Vibrio parahaemolyticus, and PAMP of LPS, but not Lipid A. A phagocytosis assay demonstrated that CgIntegrin could directly mediate phagocytosis toward bacteria as a phagocytic receptor. These results collectively suggested that CgC1qDC-5 could serve as an opsonin to recognize and bind bacteria, and subsequently interact with CgIntegrin on the hemocyte surface to enhance the CgIntegrin-mediated phagocytosis in oyster.

Persistence of antibodies 1 year after sequential administration of the 13-valent pneumococcal conjugate vaccine and the 23-valent pneumococcal polysaccharide vaccine in adults.

Vaccination with the 13-valent pneumococcal conjugate vaccine (PCV13) followed ≥ 1 year by the 23-valent pneumococcal polysaccharide vaccine (PPSV23) is recommended for immunocompetent adults ≥ 65 years of age in the United States. This study assessed antipneumococcal opsonophagocytic activity (OPA) geometric mean titers (GMTs) to PCV13 in PPSV23-naive and PPSV23-preimmunized adults 1 year after a second vaccine dose. Two parent studies were conducted previously: (1) PPSV23 vaccine-naive subjects (60-64 years of age at enrollment) received PCV13 followed by PCV13 or PPSV23 1 year later or PPSV23 followed by PCV13 1 year later; and (2) subjects (≥ 70 years of age at enrollment) vaccinated with PPSV23 ≥ 5 years before study entry received PCV13 or PPSV23 followed by PCV13 1 year later. Overall, 962 subjects (PPSV23-naive, n = 519; PPSV23-preimmunized, n = 443) who received both vaccinations in the parent studies were enrolled. Numerically higher OPA GMTs persisted for at least 1 year after administration of PCV13 as the initial vaccine (PCV13/PPSV23 or PCV13/PCV13) compared with those who received PPSV23 either 1 or 5 years prior (PPSV23/PCV13). This impairment in antibody responses to subsequent PCV13 vaccination produced by initial PPSV23 vaccination persisted for at least 1 year. OPA GMTs were numerically higher for most serotypes 1 year after 2 doses of PCV13 compared with 1 year after the first PCV13 dose. These data suggest PCV13 should be given first if both vaccines are to be administered, higher immune responses were achieved when PCV13 was given first and persisted at least 1 year (ClinicalTrials.gov Identifier: NCT01025336).