Subversion of the Complement System by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen heavily implicated in chronic diseases. Immunocompromised patients that become infected with P. aeruginosa usually are afflicted with a lifelong chronic infection, leading to worsened patient outcomes. The complement system is an integral piece of the first line of defense against invading microorganisms. Gram-negative bacteria are thought to be generally susceptible to attack from complement; however, P. aeruginosa can be an exception, with certain strains being serum resistant. Various molecular mechanisms have been described that confer P. aeruginosa unique resistance to numerous aspects of the complement response. In this review, we summarize the current published literature regarding the interactions of P. aeruginosa and complement, as well as the mechanisms used by P. aeruginosa to exploit various complement deficiencies and the strategies used to disrupt or hijack normal complement activities.

Reduced quantity and function of pneumococcal antibodies are associated with exacerbations of COPD in SPIROMICS.

While hypogammaglobulinemia is associated with COPD exacerbations, it is unknown whether frequent exacerbators have specific defects in antibody production/function. We hypothesized that reduced quantity/function of serum pneumococcal antibodies correlate with exacerbation risk in the SPIROMICS cohort. We measured total pneumococcal IgG in n = 764 previously vaccinated participants with COPD. In a propensity-matched subset of n = 200 with vaccination within five years (n = 50 without exacerbations in the previous year; n = 75 with one, n = 75 with ≥2), we measured pneumococcal IgG for 23 individual serotypes, and pneumococcal antibody function for 4 serotypes. Higher total pneumococcal IgG, serotype-specific IgG (17/23 serotypes), and antibody function (3/4 serotypes) were independently associated with fewer prior exacerbations. Higher pneumococcal IgG (5/23 serotypes) predicted lower exacerbation risk in the following year. Pneumococcal antibodies are inversely associated with exacerbations, supporting the presence of immune defects in frequent exacerbators. With further study, pneumococcal antibodies may be useful biomarkers for immune dysfunction in COPD.

Evasion of opsonization of macromolecules using novel surface-modification and biological-camouflage-mediated techniques for next-generation drug delivery.

Opsonization plays a pivotal role in hindering controlled drug release from nanoformulations due to macrophage-mediated nanoparticle destruction. While first and second-generation delivery systems, such as lipoplexes (50-150 nm) and quantum dots, hold immense potential in revolutionizing disease treatment through spatiotemporal controlled drug delivery, their therapeutic efficacy is restricted by the selective labeling of nanoparticles for uptake by reticuloendothelial system and mononuclear phagocyte system via various molecular forces, such as electrostatic, hydrophobic, and van der Waals bonds. This review article presents novel insights into surface-modification techniques utilizing macromolecule-mediated approaches, including PEGylation, di-block copolymerization, and multi-block polymerization. These techniques induce stealth properties by generating steric forces to repel micromolecular-opsonins, such as fibrinogen, thereby mitigating opsonization effects. Moreover, advanced biological methods, like cellular hitchhiking and dysopsonic protein adsorption, are highlighted for their potential to induce biological camouflage by adsorbing onto the nanoparticulate surface, leading to immune escape. These significant findings pave the way for the development of long-circulating next-generation nanoplatforms capable of delivering superior therapy to patients. Future integration of artificial intelligence-based algorithms, integrated with nanoparticle properties such as shape, size, and surface chemistry, can aid in elucidating nanoparticulate-surface morphology and predicting interactions with the immune system, providing valuable insights into the probable path of opsonization.

Functional Activities of O-Polysaccharide and Hemolysin Coregulated Protein 1 Specific Antibodies Isolated from Melioidosis Patients.

Melioidosis is a fatal tropical disease caused by the environmental Gram-negative bacterium, Burkholderia pseudomallei. This bacterium is intrinsically resistant to several antibiotics and treatment of melioidosis requires prolonged antibiotic administration. To date, there are no vaccines available for melioidosis. Previous studies have shown that humoral immunity is critical for surviving melioidosis and that O-polysaccharide (OPS) and hemolysin coregulated protein 1 (Hcp1) are important protective antigens in animal models of melioidosis. Our previous studies revealed that melioidosis patients had high levels of OPS- and Hcp1-specific antibodies and that IgG against OPS (IgG-OPS) and Hcp1 (IgG-Hcp1) were associated with patient survival. In this study, we characterized the potential function(s) of IgG-OPS and IgG-Hcp1 from melioidosis patients. IgG-OPS and IgG-Hcp1 were purified from pooled serum obtained from melioidosis patients using immuno-affinity chromatography. Antibody-dependent cellular phagocytosis assays were performed with pooled serum from melioidosis patients and compared with serum obtained from healthy controls. Serum from melioidosis patients significantly enhanced B. pseudomallei uptake into the human monocytic cell line THP-1 compared with pooled serum from healthy donors. Enhanced opsonization was observed with IgG-OPS and IgG-Hcp1 in a dose-dependent manner. Antibody-dependent complement deposition assays were performed with IgG-OPS and IgG-Hcp1 using flow cytometry and showed that there was enhanced C3b deposition on the surface of B. pseudomallei treated with IgG-OPS but to a lesser degree with IgG-Hcp1. This study provides insight into the function of IgG-OPS and IgG-Hcp1 in human melioidosis and supports that OPS and Hcp1 are potential vaccine antigens for immunization against melioidosis.

Unravelling Heterogeneities in Complement and Antibody Opsonization of Individual Liposomes as a Function of Surface Architecture.

Coating nanoparticles with poly(ethylene glycol) (PEG) is widely used to achieve long-circulating properties after infusion. While PEG reduces binding of opsonins to the particle surface, immunogenic anti-PEG side-effects show that PEGylated nanoparticles are not truly "stealth" to surface active proteins. A major obstacle for understanding the complex interplay between opsonins and nanoparticles is the averaging effects of the bulk assays that are typically applied to study protein adsorption to nanoparticles. Here, a microscopy-based method for directly quantifying opsonization at the single nanoparticle level is presented. Various surface coatings are investigated on liposomes, including PEG, and show that opsonization by both antibodies and complement C3b is highly dependent on the surface chemistry. It is further demonstrated that this opsonization is heterogeneous, with opsonized and non-opsonized liposomes co-existing in the same ensemble. Surface coatings modify the percentage of opsonized liposomes and/or opsonin surface density on the liposomes, with strikingly different patterns for antibodies and complement. Thus, this assay provides mechanistic details about opsonization at the single nanoparticle level previously inaccessible to established bulk assays.

A single-CRD C-type lectin from Haliotis discus hannai acts as pattern recognition receptor enhancing hemocytes opsonization.

C-type lectins (CTLs), as a member of the Ca2+-dependent carbohydrate recognition protein superfamily, play multiple roles in non-self recognition and the elimination of invading pathogens. In this study, a C-type lectin was identified and characterized from the Pacific abalone Haliotis discus hannai (designed as HdClec), and its open reading frame (ORF) encoded a polypeptide of 163 amino acids containing a typical signal peptide and only one carbohydrate-recognition domain (CRD). The deduced amino acid sequence of CRD in HdClec shared identities ranging from 22.4% to 39.8% with that of other identified CRDs of CTLs. A novel NPN motif was found in Ca2+-binding site 2 of HdClec. The mRNA transcripts of HdClec were detectable in all the examined tissues of non-stimulated abalones, with the highest expression in hepatopancreas (224.13-fold of that in gills). The expression of HdClec mRNA in hemocytes was significantly up-regulated after Vibrio harveyi challenge. Recombinant HdClec protein (rHdClec) could bind lipopolysaccharide (LPS) and peptidoglycan (PGN) in vitro in the presence of Ca2+. Coinciding with the PAMPs binding assay, rHdClec displayed broad agglutination activities towards Gram-negative bacteria V. splendidus, V. anguillarum, V. parahaemolyticus, V. harveyi, Escherichia coli, and Gram-positive bacteria Micrococcus luteus. Moreover, rHdClec could significantly elicit the chemotactic response of hemocytes in vitro. And the phagocytosis and encapsulation ability of hemocytes could be significantly enhanced by rHdClec. All these results showed that HdClec could function as pattern recognition receptors (PRRs) and further enhance the opsonization of hemocytes, which might play a crucial role in the innate immune responses of Pacific abalone.

Monoclonal Antibodies Opsonize Burkholderia spp. and Reduce Intracellular Actin Tail Formation in a Macrophage Infection Assay.

Melioidosis is a neglected tropical disease caused by the bacterium Burkholderia pseudomallei. The bacterium is intrinsically resistant to various antibiotics, and melioidosis is therefore difficult to treat successfully without a relapse in infection. B. pseudomallei is an intracellular pathogen and therefore, to eradicate the infection, antimicrobials must be able to access bacteria in an intracellular niche. This study assessed the ability of a panel of monoclonal antibodies (MAbs) to opsonize Burkholderia species and determine the effect that each antibody has on bacterial virulence in vitro. Murine macrophage infection assays demonstrated that monoclonal antibodies against the capsule of B. pseudomallei are opsonizing. Furthermore, one of these monoclonal antibodies reduced bacterial actin tail formation in our in vitro assays, indicating that antibodies could reduce the intracellular spread of Burkholderia thailandensis. The data presented in this paper demonstrate that monoclonal antibodies are opsonizing and can decrease bacterial actin tail formation, thus decreasing their intracellular spread. These data have informed selection of an antibody for development of an antibody-antibiotic conjugate (AAC) for melioidosis. IMPORTANCE Melioidosis is difficult to treat successfully due to the causal bacterium being resistant to many classes of antibiotics, therefore limiting available therapeutic options. New and improved therapies are urgently required to treat this disease. Here, we have investigated the potential of monoclonal antibodies to target this intracellular pathogen. We have demonstrated that monoclonal antibodies can target the bacterium, increase uptake into macrophages, and reduce actin tail formation required by the bacterium for spread between cells. Through targeting the bacterium with antibodies, we hope to disarm the pathogen, reducing the spread of infection. Ultimately, we aim to use an opsonizing antibody to deliver antibiotics intracellularly by developing an antibody-antibiotic conjugate therapeutic for melioidosis.

Analysis of complement deposition and processing on Chlamydia trachomatis.

Chlamydia trachomatis (C. trachomatis) is the leading cause of sexually transmitted bacterial infections worldwide, with over 120 million annual cases. C. trachomatis infections are associated with severe reproductive complications in women such as extrauterine pregnancy and tubal infertility. The infections are often long lasting, associated with immunopathology, and fail to elicit protective immunity which makes recurrent infections common. The immunological mechanisms involved in C. trachomatis infections are only partially understood. Murine infection models suggest that the complement system plays a significant role in both protective immunity and immunopathology during primary Chlamydia infections. However, only limited structural and mechanistic evidence exists on complement-mediated immunity against C. trachomatis. To expand our current knowledge on this topic, we analyzed global complement deposition on C. trachomatis using comprehensive in-depth mass spectrometry-based proteomics. We show that factor B, properdin, and C4b bind to C. trachomatis demonstrating that C. trachomatis-induced complement activation proceeds through at least two activation pathways. Complement activation leads to cleavage and deposition of C3 and C5 activation products, causing initiation of the terminal complement pathway and deposition of C5b, C6, C7, C8, C9 on C. trachomatis. Interestingly, using immunoelectron microscopy, we show that C5b-9 deposition occurred sporadically and only in rare cases formed complete lytic terminal complexes, possibly caused by the presence of the negative regulators vitronectin and clusterin. Finally, cleavage analysis of C3 demonstrated that deposited C3b is degraded to the opsonins iC3b and C3dg and that this complement opsonization facilitates C. trachomatis binding to human B-cells.

Characterization and protective activity of monoclonal antibodies directed against Fe (3+) ABC transporter substrate-binding protein of Glaesserella parasuis.

Glässer's disease is caused by the agent Glaesserella parasuis and is difficult to prevent and control. Candidate screening for subunit vaccines contributes to the prevention of this disease. Therefore, in this study, the inactivated G. parasuis reference serovar 5 strain (G. parasuis-5) was used to generate specific monoclonal antibodies (mAbs) to screen subunit vaccine candidates. Six mAbs (1A12, 3E3, 4C6, 2D1, 3E6, and 4B2) were screened, and they all reacted with the G. parasuis serovar 5 strain according to laser confocal microscopy and flow cytometry (FCM). Indirect enzyme-linked immunosorbent assay (ELISA) showed that one mAb 2D1, can react with all 15 reference serovars of G. parasuis. Protein mass spectrometry and Western blot analysis demonstrated that mAb 2D1 specifically reacts with Fe (3+) ABC transporter substrate-binding protein. A complement killing assay found that the colony numbers of bacteria were significantly reduced in the G. parasuis-5 group incubated with mAb 2D1 (p < 0.01) in comparison with the control group. Opsonophagocytic assays demonstrated that mAb 2D1 significantly enhanced the phagocytosis of 3D4/21 cells by G. parasuis (p < 0.05). RAW264.7 cells with stronger phagocytic ability were also used for the opsonophagocytic assay, and the difference was highly significant (p < 0.01). Passive immunization of mice revealed that mAb 2D1 can eliminate the bacteria in the blood and provide protection against G. parasuis-5. Our study found one mAb that can be used to prevent and control G. parasuis infection in vivo and in vitro, which may suggest that Fe (3+) ABC transporter substrate-binding protein is an immunodominant antigen and a promising candidate for subunit vaccine development.

[Electronic microscopy assessment of immunological disorders in the secret of the prostate in patients with chronic recurrent bacterial prostatitis].

INTRODUCTION: Chronic recurrent bacterial prostatitis (CRPD) is an urgent problem of modern urology and andrology. OBJECTIVE: To study the immunological features of the secretion of the prostate by electron microscopy in patients with chronic recurrent bacterial prostatitis. MATERIALS AND METHODS: The analysis of the morphometric study of neutrophils in the secretion of the prostate was carried out in 90 patients with chronic bacterial prostatitis, who were divided into two groups. Group I (study) (n=51) with chronic recurrent bacterial prostatitis (CRBP) and group II (control) (n=39) with chronic primary diagnosed bacterial prostatitis (CPDBP). RESULTS: At electron microscopy of ALE in most patients with CRBP of the group, the cytological picture of ALE was represented by inactive neutrophils with pathology of phagocytosis. The cells are of the correct rounded shape, without pseudopodia, with light cytoplasm. The cytological picture of ALE in CPDBP is characterized by the absence of impaired local immunity. The normal process of phagocytosis is recorded, where many pseudopodia of the segmented neutrophil completely complete the process of opsonization of microorganisms in the area of the inflammatory process. CONCLUSIONS: In patients with CRBP, in 100% of cases, dysfunction of immunocompetent prostate cells was noted, which is the basis for the appointment of immunoactive therapy for CRBP.

Opsonophagocytosis of Chlamydia pneumoniae by Human Monocytes and Neutrophils.

The human respiratory tract pathogen Chlamydia pneumoniae, which causes mild to severe infections, has been associated with the development of chronic inflammatory diseases. To understand the biology of C. pneumoniae infections, several studies have investigated the interaction between C. pneumoniae and professional phagocytes. However, these studies have been conducted under nonopsonizing conditions, making the role of opsonization in C. pneumoniae infections elusive. Thus, we analyzed complement and antibody opsonization of C. pneumoniae and evaluated how opsonization affects chlamydial infectivity and phagocytosis in human monocytes and neutrophils. We demonstrated that IgG antibodies and activation products of complement C3 and C4 are deposited on the surface of C. pneumoniae elementary bodies when incubated in human serum. Complement activation limits C. pneumoniae infectivity in vitro and has the potential to induce bacterial lysis by the formation of the membrane attack complex. Coculture of C. pneumoniae and freshly isolated human leukocytes showed that complement opsonization is superior to IgG opsonization for efficient opsonophagocytosis of C. pneumoniae in monocytes and neutrophils. Neutrophil-mediated phagocytosis of C. pneumoniae was crucially dependent on opsonization, while monocytes retained minor phagocytic potential under nonopsonizing conditions. Complement opsonization significantly enhanced the intracellular neutralization of C. pneumoniae in peripheral blood mononuclear cells and neutrophils and almost abrogated the infectious potential of C. pneumoniae In conclusion, we demonstrated that complements limit C. pneumoniae infection in vitro by interfering with C. pneumoniae entry into permissive cells by direct complement-induced lysis and by tagging bacteria for efficient phagocytosis in both monocytes and neutrophils.

Antibody Opsonization Enhances Early Interactions between Yersinia pestis and Neutrophils in the Skin and Draining Lymph Node in a Mouse Model of Bubonic Plague.

Bubonic plague results when Yersinia pestis is deposited in the skin via the bite of an infected flea. Bacteria then traffic to the draining lymph node (dLN) where they replicate to large numbers. Without treatment, this infection can result in highly fatal septicemia. Several plague vaccine candidates are currently at various stages of development, but no licensed vaccine is available in the United States. Though polyclonal and monoclonal antibodies (Ab) can provide complete protection against bubonic plague in animal models, the mechanisms responsible for this antibody-mediated immunity (AMI) to Y. pestis remain poorly understood. Here, we examine the effects of Ab opsonization on Y. pestis interactions with phagocytes in vitro and in vivo Opsonization of Y. pestis with polyclonal antiserum modestly increased phagocytosis/killing by an oxidative burst of murine neutrophils in vitro Intravital microscopy (IVM) showed increased association of Ab-opsonized Y. pestis with neutrophils in the dermis in a mouse model of bubonic plague. IVM of popliteal LNs after intradermal (i.d.) injection of bacteria in the footpad revealed increased Y. pestis-neutrophil interactions and increased neutrophil crawling and extravasation in response to Ab-opsonized bacteria. Thus, despite only having a modest effect in in vitro assays, opsonizing Ab had a dramatic effect in vivo on Y. pestis-neutrophil interactions in the dermis and dLN very early after infection. These data shed new light on the importance of neutrophils in AMI to Y. pestis and may provide a new correlate of protection for evaluation of plague vaccine candidates.

Quorum-sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization.

When Drosophila melanogaster feeds on Pseudomonas aeruginosa, some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum-sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI-independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester-containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4-mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild-type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense.

Precision Nanomedicine Development Based on Specific Opsonization of Human Cancer Patient-Personalized Protein Coronas.

When a nanomedicine is administrated into the human body, biomolecules in biological fluids, particularly proteins, form a layer on the surface of the nanoparticle known as a "personalized protein corona". An understanding of the formation and behavior of the personalized protein corona not only benefits the nanotherapy treatment efficacy but also can aid in disease diagnosis. Here we used Gd@C82(OH)22 nanoparticles, a nanomedicine effective against several types of cancer, as a model nanomedicine to investigate the natural protein fingerprint of the personalized protein corona formed in 10 human lung squamous cell carcinoma patients. Our analysis revealed a specific biomarker, complement component C1q, in lung cancer personalized protein coronas, abundantly bound to Gd@C82(OH)22 NPs. This binding altered the secondary structure of C1q protein and led to the activation of an innate immune response, which could be exploited for cancer immune therapy. On the basis of this finding, we provide a new strategy for the development of precision nanomedicine derived from opsonization of a unique protein fingerprint within patients. This approach overcomes the common pitfall of protein corona formation and exploits the corona proteins to generate a precision nanomedicine and diagnostic tool.

Pentraxins in the activation and regulation of innate immunity.

Humoral fluid phase pattern recognition molecules (PRMs) are a key component of the activation and regulation of innate immunity. Humoral PRMs are diverse. We focused on the long pentraxin PTX3 as a paradigmatic example of fluid phase PRMs. PTX3 acts as a functional ancestor of antibodies and plays a non-redundant role in resistance against selected microbes in mouse and man and in the regulation of inflammation. This molecule interacts with complement components, thus modulating complement activation. In particular, PTX3 regulates complement-driven macrophage-mediated tumor progression, acting as an extrinsic oncosuppressor in preclinical models and selected human tumors. Evidence collected over the years suggests that PTX3 is a biomarker and potential therapeutic agent in humans, and pave the way to translation of this molecule into the clinic.

Antibody opsonization enhances MAIT cell responsiveness to bacteria via a TNF-dependent mechanism.

Mucosal-associated invariant T (MAIT) cells are an abundant human T-cell subset with antimicrobial properties. They can respond to bacteria presented via antigen-presenting cells (APCs) such as macrophages, which present bacterially derived ligands from the riboflavin synthesis pathway on MR1. Moreover, MAIT cells are also highly responsive to cytokines which enhance and even substitute for T-cell receptor-mediated signaling. The mechanisms leading to an efficient presentation of bacteria to MAIT cells by APCs have not been fully elucidated. Here, we showed that the monocytic cell line THP-1 and B cells activated MAIT cells differentially in response to Escherichia coli. THP-1 cells were generally more potent in inducing IFNγ and IFNγ/TNF production by MAIT cells. Furthermore, THP-1, but not B, cells produced TNF upon bacterial stimulation, which in turn supported IFNγ production by MAIT cells. Finally, we addressed the role of antibody-dependent opsonization of bacteria in the activation of MAIT cells using in vitro models. We found that opsonization had a substantial impact on downstream MAIT cell activation by monocytes. This was associated with enhanced activation of monocytes and increased TNF release. Importantly, this TNF acted in concert with other cytokines to drive MAIT cell activation. These data indicate both a significant interaction between adaptive and innate immunity in the response to bacteria, and an important role for TNF in MAIT cell triggering.

Neutrophil-mediated antifungal activity against highly virulent Cryptococcus gattii strain R265.

Vaccine-induced immune responses, including neutrophil, macrophage, and T-cell responses, ameliorate cryptococcosis caused by Cryptococcus gattii. However, whether neutrophils can exert fungicidal activity against C. gattii remains to be elucidated. Therefore, in this study, we investigated the neutrophil-mediated fungicidal effect against C. gattii R265 in vitro and compared it to the related fungal pathogen, Cryptococcus neoformans standard strain H99. We found that neutrophils recognized, phagocytosed, and killed C. gattii R265 in the presence of fresh mouse serum. This antifungal effect required phagocytosis and serine protease activity but not nicotinamide adenine dinucleotide phosphate oxidase activity. We also demonstrated that C. gattii R265 was more resistant to oxidative and nitrosative stress than C. neoformans H99. Together, these findings indicate that neutrophils can exert fungicidal activity against highly virulent C. gattii, at least under in vitro conditions.

Functional characterization of a mannose-binding lectin (MBL) from Nile tilapia (Oreochromis niloticus) in non-specific cell immunity and apoptosis in monocytes/macrophages.

Mannose-binding lectin (MBL), a soluble pattern recognition receptor, is able to recognize antigen and participate in non-specific cell immunity, such as regulation of inflammation, migration, opsonization, phagocytosis and killing, which plays an important role in innate immunity. In this study, we have investigated the contributing mechanisms and effects of MBL on the cell immunity of Nile tilapia (Oreochromis niloticus) monocytes/macrophages. The mRNA expression level of OnMBL was significantly up-regulated in monocytes/macrophages after in vitro bacterial infection (Streptococcus agalactiae and Aeromonas hydrophila). Recombinant OnMBL ((r)OnMBL) protein could participate in the regulation of inflammation, migration, and enhancement of phagocytosis and respiratory burst activity in monocytes/macrophages. Moreover, the (r)OnMBL could induce the apoptosis of monocytes/macrophages. Taken together, the results of this study indicated that OnMBL is likely to involve in immune regulation, which may play an important role in host defense of innate immunity in Nile tilapia.

Identification and characterization of a B-type mannose-binding lectin from Nile tilapia (Oreochromis niloticus) in response to bacterial infection.

Lectins are a group of carbohydrate-binding proteins, which play an important role in innate immune system against pathogen infection. In this study, a B-type mannose-binding lectin (OnBML) was identified from Nile tilapia (Oreochromis niloticus), and characterized at expression patterns against bacterial infection and capability to promote phagocytosis by macrophages. The open reading frame of OnBML is 354 bp of nucleotide sequence encoding polypeptides of 117 amino acids. The deduced protein is highly homologous to other teleost BMLs, containing two repeats of the conserved mannose-binding motif QXDXNXVXY. Expression of OnBML was widely exhibited in all examined tissues, with the most abundance in spleen and following gill, peripheral blood, and head kidney. The OnBML expressions were significantly up-regulated following two major bacterial infections including a Gram-positive bacterium (Streptococcus agalactiae) and a Gram-negative bacterium (Aeromonas hydrophila) in vivo and in vitro. Recombinant OnBML protein possessed capacities of mannose-binding and calcium-dependent agglutination to S. agalactiae and A. hydrophila, and promoted the phagocytosis by macrophages. Taken together, the present study indicated that OnBML is likely to get involved in host defense against bacterial infection in Nile tilapia.

Self-assembly of trimethyl chitosan and poly(anionic amino acid)-peptide antigen conjugate to produce a potent self-adjuvanting nanovaccine delivery system.

Short peptides derived from virulent pathogen proteins are promising antigens for the development of vaccines against infectious diseases. However, in order to mimic the danger signals associated with natural infection and stimulate an adaptive immune response, peptide antigens must be co-delivered with immune adjuvants. In this study, a group A streptococcus (GAS) M-protein derived B-cell epitope: J8, and universal T-helper epitope P25 containing peptides, were chemically coupled with different anionic amino acid-based polymers. The poly(anionic amino acid)-peptide antigen conjugates were mixed with trimethyl chitosan (TMC) to produce self-adjuvanting nanoparticulate vaccine candidates. TMC from two different sources were used to analyse their effect on immunogenicity. The nanoparticles produced from a peptide modified with 10 residues of polyglutamic acid and fungal TMC (NP5) stimulated production of the highest levels of serum antibodies in outbred mice. These antibodies were opsonic against all clinical GAS isolates tested.