Opsonophagocytosis Assay Research Articles

Genome sequences of 36 Streptococcus pneumoniae strains optimized for the multiplexed opsonophagocytosis killing assay.

A multiplexed opsonophagocytosis assay (MOPA) was developed as a cost-effective, high-throughput biological assay to evaluate the efficacy of pneumococcal vaccines by in vitro measurement of opsonophagocytic activity of anti-capsular antibodies. Here, we report draft genomes of the 36 strains of Streptococcus pneumoniae developed for use in the reference pneumococcal MOPA.

Current challenges and improvements in assessing the immunogenicity of bacterial vaccines.

The increase in antimicrobial-resistant bacterial strains has highlighted the need for a new vaccine strategy. The primary goal of a candidate vaccine is to prevent disease, by inducing a persistent immunologic memory, through the activation of pathogen-specific immune response. Antibody titer is the main parameter used to assess the immunogenicity of bacterial vaccine candidates and it is the most widely used as a correlate of protection. On the other hand, the antibody titer alone cannot provide complete information on all the activity mediated by antibodies which can only be assessed by functional assays, like the serum bactericidal assay and the opsonophagocytosis assay. However, due to the involvement of many biological factors, these assays are difficult to standardize. Some improvements have been achieved in recent years, but further optimizations are needed to minimize inter- and intra-laboratories variability and to allow the applicability of these functional assays for the vaccine immunogenicity assessment on a larger scale.

Deep-learning image analysis for high-throughput screening of opsono-phagocytosis-promoting monoclonal antibodies against Neisseria gonorrhoeae

Antimicrobial resistance (AMR) is nowadays a global health concern as bacterial pathogens are increasingly developing resistance to antibiotics. Monoclonal antibodies (mAbs) represent a powerful tool for addressing AMR thanks to their high specificity for pathogenic bacteria which allows sparing the microbiota, kill bacteria through complement deposition, enhance phagocytosis or inhibit bacterial adhesion to epithelial cells. Here we describe a visual opsono-phagocytosis assay which relies on confocal microscopy to measure the impact of mAbs on phagocytosis of the bacterium Neisseria gonorrhoeae by macrophages. With respect to traditional CFU-based assays, generated images can be automatically analysed by convolutional neural networks. Our results demonstrate that confocal microscopy and deep learning-based analysis allow screening for phagocytosis-promoting mAbs against N. gonorrhoeae, even when mAbs are not purified and are expressed at low concentration. Ultimately, the flexibility of the staining protocol and of the deep-learning approach make the assay suitable for other bacterial species and cell lines where mAb activity needs to be investigated.

Invasive and Non-invasive Clinical Haemophilus influenzae Type A Isolates Activate Differentiated HL-60 Cells In Vitro.

The effective elimination of encapsulated bacteria like Haemophilus influenzae type a (Hia) relies on immune mechanisms such as complement-mediated opsonophagocytosis by neutrophils in coordination with opsonization by anti-capsular antibodies. This study evaluated if Hia could activate the immune response through neutrophils and if these responses differed between encapsulated versus unencapsulated or invasive versus non-invasive strains. HL-60-derived neutrophil-like cells (dHL-60), differentiated with 1.25% dimethyl sulfoxide over 9 days, were used in an opsonophagocytosis assay and in vitro infection model to measure Hia's susceptibility to killing and dHL-60 surface molecule expression, respectively. The impact of strain-specific features on the immune response was investigated using clinical isolates of a dominant North American sequence type (ST)-23, including Hia 11-139 (encapsulated, invasive), 14-61 (encapsulated, non-invasive), 13-0074 (unencapsulated, invasive), as well as a representative ST-4 isolate (Hia 13-240, encapsulated, invasive), and a nontypeable strain (NTHi 375, unencapsulated, non-invasive). Unencapsulated and non-invasive Hi strains were more susceptible to killing by the innate immune response while the ST-23 invasive strain, Hia 11-139 required serum antibodies for destruction. Flow cytometry analysis showed increased expression of co-stimulatory molecule ICAM-1 and Fc receptors (CD89, CD64) but decreased expression of the Fc receptor CD16, revealing potential mechanisms of neutrophil-mediated defense against Hia that extend to both non-invasive and invasive strains. Hia clinical isolates with diverse pathogenicity illustrated contrasting susceptibility to killing by immune mechanisms while maintaining the same capacity to activate neutrophil-like cells, further underscoring the need for additional studies on Hia's pathogenesis.

Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326).

Syphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Tp's repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of Tp by rabbit peritoneal macrophages. Three overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed "Epivolve" (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin scaffold (PfTrxBamA/ECL4). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (e.g., opsonization) and validate the mouse assay. Sera from Tp-infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using PfTrxBamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays. Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of PfTrxBamA/ECL4. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope. Epivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by Tp infection.

The Multiplexed Opsonophagocytosis Killing Assay (MOPA) in Evaluating Specific Antibody Responses in Patients with Recurrent Infections

Assessments of antibody responses to vaccines are essential tools for evaluating humoral immune responses in subjects with suspected inborn errors of immunity (IEI). Low pneumococcal IgG levels following pneumococcal vaccination indicate suboptimal humoral immune function and are often used for diagnosis of IEI. The Multiplexed Opsonophagocytosis Assay (MOPA) measures pneumococcal antibody function (PAF) via killing of pneumococci by serum antibodies in vitro. MOPA may also be useful for clinical diagnosis but has not been studied in patients with suspected IEI. We aimed to compare PAF and Streptococcus pneumoniae IgG levels in patients with recurrent infections. We also evaluated the clinical history and other immune workup results, when available. We collected serum samples from n = 17 patients with a history of recurrent infections undergoing pneumococcal IgG measurements by their primary clinical immunologists. Baseline sera (prior to vaccine challenge) were analyzed for PAF, for the serotypes present in pneumococcal conjugate vaccine (PCV13), and S. pneumoniae 23 serotypes IgG levels using MOPA and a multiplexed bead array assay, respectively. The cohort includes subjects from 2 to 60 years of age, 53% females, 94% Caucasian, and 30% Hispanic. Preliminary results show that 76% patients had protective PAF for 85% or more of the tested serotypes. Of these, 69% patients had received a pneumococcal vaccine in the previous five years as an indication for an underlying high risk medical condition or as part of their immunization schedule. Protective results for serotypes 3, 4, 9V, and 18C were discrepant between IgG levels and PAF in more than 50% of patients. We also found discrepancy between PAF and antibody levels in 35% of patients, showing intact PAF for 5 or more serotypes despite low serotype IgG levels; 83% of whom had no significant sinopulmonary infections in the last year. MOPA analysis between pre and post pneumococcal vaccine administration, in those patients for which it is clinically indicated, is currently in process. This analysis may provide more information regarding the diagnostic performance and predictive value of MOPA in patients with suspected IEI, but preliminary data shows that MOPA might be more reflective of protection than S. pneumonia IgG levels.

Sublingual immunisation with GBS serotype III capsular polysaccharide-tetanus toxoid conjugate vaccine induces systemic and mucosal antibody responses which are opsonophagocytic and inhibit GBS colonisation of vaginal epithelial cells

No vaccines are currently licensed against Group B streptococcus (GBS), an important cause of morbidity and mortality in babies and adults. Using a mouse model, and in vitro opsonophagocytosis and colonisation assays, we evaluated the potential of a sublingually-administered polysaccharide-conjugate vaccine against GBS serotype III. Sublingual immunisation of mice with 10 µg of GBS conjugate vaccine once a week for 5 weeks induced a substantial systemic IgG anti-polysaccharide response which was similar to the level induced by subcutaneous immunsation. In addition, sublingual immunisation also induced mucosal (IgA) antibody responses in the mouth, intestines and vagina. Immune sera and intestinal washes were functionally active at mediating killing of the homologous GBS serotype III in an opsonophagocytosis assay. In addition, intestinal and vaginal washes inhibited the colonisation of mouse vaginal epithelial cells by the vaccine homologous strain. These results suggest that, in addition to the induction of high levels of IgG antibodies that could be transduced from the immunised mother to the foetus to protect the newborn against GBS infection, sublingual immunisation can elicit a substantial mucosal antibody response which might play an important role in the prevention of GBS colonisation in immunised women, thereby eliminating the risk of GBS transmission from the mother to the baby during pregnancy or at birth.

Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease.

Streptococcus pneumoniae colonizes the nasopharynx asymptomatically but can also cause severe life-threatening disease. Importantly, stark differences in carbohydrate availability exist between the nasopharynx and invasive disease sites, such as the bloodstream, which most likely impact S. pneumoniae's behavior. Herein, using chemically defined medium (CDM) supplemented with physiological levels of carbohydrates, we examined how anatomical site-specific carbohydrate availability impacted S. pneumoniae physiology and virulence. S. pneumoniae cells grown in CDM modeling the nasopharynx (CDM-N) had reduced metabolic activity and a lower growth rate, demonstrated mixed acid fermentation with marked H2O2 production, and were in a carbon-catabolite repression (CCR)-derepressed state versus S. pneumoniae cells grown in CDM modeling blood (CDM-B). Using transcriptome sequencing (RNA-seq), we determined the transcriptome for the S. pneumoniae wild-type (WT) strain and its isogenic CCR-deficient mutant in CDM-N and CDM-B. Genes with altered expression as a result of changes in carbohydrate availability or catabolite control protein deficiency, respectively, were primarily involved in carbohydrate metabolism, but also encoded established virulence determinants, such as polysaccharide capsule and surface adhesins. We confirmed that anatomical site-specific carbohydrate availability directly influenced established S. pneumoniae virulence traits. S. pneumoniae cells grown in CDM-B formed shorter chains, produced more capsule, were less adhesive, and were more resistant to macrophage killing in an opsonophagocytosis assay. Moreover, growth of S. pneumoniae in CDM-N or CDM-B prior to the challenge of mice impacted relative fitness in a colonization model and invasive disease model, respectively. Thus, anatomical site-specific carbohydrate availability alters S. pneumoniae physiology and virulence, in turn promoting anatomical site-specific fitness.

A phase 3 clinical trial of MINHAI PCV13 in Chinese children aged from 7 months to 5 years old

BackgroundPneumococcus lead to various kinds of invasive disease such as pneumonia, otitis media, meningitis, bacteremia and so on. It has been a great threat to children under 5. A new 13-valent pneumococcal conjugate vaccine (PCV13) with carrier tetanus toxoid and diphtheria toxoid was developed by MINHAI, aiming to prevent pneumococcus infection. In this study, we reported the safety and immunogenicity of MINHAI PCV13 in Chinese children aged from 7 months to 5 years old. MethodsA randomized, double-blinded, parallelized phase III clinical trial was operated in 900 participants. Haemophilus influenzae type B conjugate vaccine (Hib) served as negative control. PCV13 and Hib were intramuscular injected to participants at a ratio of 2:1. Local and systemic adverse events (AEs) and severe adverse events (SAEs) were recorded to evaluate the safety of PCV13. Blood samples were collected before and after immunization for the detecting of serotype-specific anti-polysaccharide immunoglobulin (Ig)G and opsonophagocytosis assay (OPA). The proportion of IgG concentration ≥ 0.35 μg/mL (IgG positive rate), IgG geometric mean concentration (GMC), OPA geometric mean titer (GMT), and other indicators were analyzed to evaluate the immunogenicity of PCV13. ResultsDuring the study period, no PCV13 associated SAE happened. Incidences of several AEs in PCV13 groups were higher than the Hib groups, but most of them were mild or moderate. For all 13 serotypes, IgG and OPA indicators of the PCV13 groups were generally superior to the Hib groups, and the differences were mostly statistically significant, which indicates that MINHAI PCV13 can effectively induce pneumococcal specific antibody. ConclusionThe study demonstrates that MINHAI PCV13 has sufficient immunogenicity and safety in Chinese children aged from 7 months to 5 years old.Clinical trial registration: NCT02494999.

Multicomponent Vaccines against Group A Streptococcus Can Effectively Target Broad Disease Presentations

Group A Streptococcus (GAS) is an important global human pathogen, with a wide range of disease presentations, from mild mucosal infections like pharyngitis to invasive diseases such as toxic shock syndrome. The effect on health and mortality from GAS infections is substantial worldwide, particularly from autoimmune sequelae-like rheumatic heart disease (RHD), and there is currently no licenced vaccine. We investigated protein antigens targeting a broad range of GAS disease presentations as vaccine components in individual and combination formulations. The potency and functional immunity generated were evaluated and compared between groups. Antibodies against all components were found in pooled human IgG (IVIG) and an immune response generated following the subcutaneous immunisation of mice. A combination immunisation showed a reduction in IgG response for SpyCEP but an increase for Cpa and Mac-1 (IdeS). An opsonophagocytosis assay (OPA) showed the killing of GAS with immune sera against M protein and combination groups, with a lower killing activity observed for immune sera against other individual antigens. Specific antigen assays showed functional immunity against SpyCEP and Mac-1 from both individual and combination immunisations, with the activity correlating with antibody titres. However, efficient blocking of the binding activity of Cpa to collagen I and fibronectin could not be demonstrated with immune sera or purified IgG. Our data indicate that combination immunisations, while effective at covering a broader range of virulence factors, can also affect the immune response generated. Further, our results showed that an OPA alone is inadequate for understanding protection from vaccination, particularly when considering protection from immune evasion factors and evaluation of the colonisation leading to pharyngitis.

Anti-flagellin IgY antibodies protect against Pseudomonas aeruginosa infection in both acute pneumonia and burn wound murine models in a non-type-specific mode

Pseudomonas aeruginosa (PA) is one of the most dominant causes of nosocomial infections in burn patients. Increasing emergence of antibiotic-resistant strains highlights the need for novel antimicrobial agents. Flagellin, the main component protein of flagellum, is determined as the major antigen interacting with anti-P. aeruginosa IgY antibodies. The current study was aimed to evaluate the antibacterial potency of IgY antibodies raised against recombinant type A, and B flagellins. The immunogenicity and specificity of IgY antibodies were confirmed through indirect ELISA and western blot analysis, respectively. Anti-flagellin IgYs reduced the motility, biofilm formation and invasion potency of both strains. The cell surface hydrophobicity (CSH) of bacteria was increased upon IgY treatment, and in vitro opsonophagocytosis assay confirmed the high protective potency of specific antibodies via polymorphonuclear leukocyte (PMN)-augmented bacterial cell killing. The protective efficacy of IgYs was also studied in both acute pneumonia and burn wound murine models. Anti-flagellin B-IgY induced 100 % and 40 % protection against laboratory, and hospital strains in burn wound model, respectively. Protection in acute pneumonia against all strains was 100 %. Anti-flagellin A-IgY failed to protect mice in burn wound model, but provided 100 % protection against all strains in acute pneumonia challenge. In vitro, ex vivo and in vivo experiments confirmed the dose-dependent and non-type specific essence of anti-flagellin IgY antibodies, providing the benefit of covering all strain types in a dose dependent manner. Our findings provide evidence that anti-flagellin IgY antibodies qualify as novel economical therapeutic option against PA infection.

Immunization against Pseudomonas aeruginosa using Alg-PLGA nano-vaccine.

Objective(s): Pseudomonas aeruginosa is the bacterium that causes of pulmonary infection among chronically hospitalized patients. Alginate is a common surface antigen of P. aeruginosa with a constant structure that which makes it an appropriate target for vaccines. In this study, P. aeruginosa alginate was conjugated with to PLGA nanoparticles, and its immunogenicity was characterized as a vaccine.Materials and Methods:Alginate was isolated from a mucoid strain of P. aeruginosa and conjugated with to PLGA with˝ N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride ˝= ˝EDAC˝ and N-Hydroxysuccinimide (NHS). Chemical characterization of prepared nano-vaccine was performed using FTIR Spectroscopy, Zetasizer, and Atomic Force Microscopy (AFM). The immunogenicity of this nano-vaccine was evaluated through intramuscular injection into BALB/c mice. Four groups of mice were subjected to the injection of alginate–PLGA, and two weeks after the last administration step, opsonophagocytosis assay, IgG detection, challenge, and cytokine determination via ELISA were carried out.Results:Alginate-PLGA conjugation was corroborated by FTIR, Zetasizer, and AFM. The ELISA consequence showed that alginate was prospering in the instigation of the humoral immunity.The immunogenicity enhanced against the alginate-PLGA. Remarkably diminished bacterial titer in the spleen of the immunized mice posterior to challenge with PAO1 strain in comparison with the alginate alone and control groups.Conclusion:The bacterial burden in the spleen significantly decreased after the challenge (P<0.05). The opsonic activity was significantly increased in the alginate- PLGA group (P<0.05).

Immunological effects of two new nanovaccines against Brucella based on OPS and LPS antigens conjugated with PLGA nanoparticles

The risk of catching brucellosis and the absence of effective human vaccines has led to the use of new strategies to produce new vaccines against infections caused by this bacterium. In this study, the effect of two new designed nanovaccines on the production of immunoglobulins and their proper functions was investigated. Firstly, the production of immunoglobulins in the serum of the mouse model after three vaccination periods with lipopolysaccharide and oligopolysaccharide antigens in conjugation with PLGA nanoparticles were studied. The mice were divided into 6 groups and each group received LPS-PLGA, OPS-PLGA, LPS, OPS, PLGA and PBS separately. Immunoglobulin titers were measured every two weeks after each vaccination period. Two weeks after the last vaccination, opsonophagocytosis test were performed to evaluate the efficacy and function of produced immunoglobulins. In order to investigate the protective effect of nanovaccines in vivo, the mice were challenged with Brucella abortus bacteria. Immune responses have increased on immunoglobulin production after each vaccination period. The results of the opsonophagocytosis assay showed that produced immunoglobulins have the superior performance to antibodies produced against the pure antigens. By performing the challenge assay, it was found that the immune system of mice receiving new nanovaccines were able to remove more bacteria from the body of mice. In general, it can be concluded that both new prepared nanovaccines can be considered to improve the immunization process in animals and humans against brucellosis although LPS-PLGA conjugate creates more immunogenicity compared to OPS-PLGA nanovaccine.

Immunogenicity Comparison of a Next Generation Pneumococcal Conjugate Vaccine in Animal Models and Human Infants.

Evaluation of a pneumococcal conjugate vaccine (PCV) in an animal model provides an initial assessment of the performance of the vaccine prior to evaluation in humans. Cost, availability, study duration, cross-reactivity and applicability to humans are several factors which contribute to animal model selection. PCV15 is an investigational 15-valent PCV which includes capsular polysaccharides from pneumococcal serotypes (ST) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F all individually conjugated to cross-reactive material 197 (CRM197). Immunogenicity of PCV15 was evaluated in infant rhesus macaques (IRM), adult New Zealand white rabbits (NZWR) and CD1 mice using multiplexed pneumococcal electrochemiluminescent (Pn ECL) assay to measure serotype-specific IgG antibodies, multiplexed opsonophagocytosis assay (MOPA) to measure serotype-specific functional antibody responses and bacterial challenge in mice to evaluate protection against a lethal dose of S. pneumoniae. PCV15 was immunogenic and induced both IgG and functional antibodies to all 15 vaccine serotypes in all animal species evaluated. PCV15 also protected mice from S. pneumoniae serotype 14 intraperitoneal challenge. Opsonophagocytosis assay (OPA) titers measured from sera of human infants vaccinated with PCV15 in a Phase 2 clinical trial showed a good correlation with that observed in IRM (rs=0.69, P=0.006), a medium correlation with that of rabbits (rs=0.49, P=0.06), and no correlation with that of mice (rs=0.04, P=0.89). In contrast, there was no correlation in serum IgG levels between human infants and animal models. These results demonstrate that PCV15 is immunogenic across multiple animal species, with IRM and human infants showing the best correlation for OPA responses.

Assessment of PhtD C-Terminal Immunogenicity by Opsonophagocytosis Assay (OPA) with OMVs as Adjuvants

Assessment of PhtD C-Terminal Immunogenicity by Opsonophagocytosis Assay (OPA) with OMVs as Adjuvants

2710. Novel Analytical Models for Pneumococcal Multiplex Opsonophagocytosis Assay Results from a Healthy Older Adult Population Vaccinated with PCV13

BackgroundThe multiplexed opsonophagocytosis assay (MOPA) measures killing of pneumococci by serum antibodies, and is the primary method for measuring pneumococcal antibodies in adults. However, pre-vaccine opsonic activity and vaccine response are highly variable among serotypes and individuals, and there are currently no criteria to define normal MOPA results.MethodsWe performed post-hoc analysis of data from n = 311 healthy, pneumococcal-vaccine naïve adults aged 55–74 who received 0.5 mL PCV13, and had MOPA performed for PCV13 serotypes (except serotype 3) at baseline, then on days 29 and 181 post-vaccine (Jackson et al. 2018, Vaccine). MOPA results (reported as opsonic index, or OI) were standardized using pneumococcal reference serum 007sp. Pairwise comparisons of proportions of undetectable baseline OI (≤ 4) between serotypes were performed using Pearson’s Chi-square. Immunogenicity (mean change in OI at day 29 post-PCV among samples with undetectable baseline OI) was compared between serotypes using one-way ANOVA. We then assigned a score based on cutoffs for pre-vaccine OI (cutoff 1, or C1) and fold-rise in OI at day 29 (cutoff 2, or C2) for each serotype, as shown in Figure 1. The sum of the scores for 12 serotypes was determined for each participant. We plotted the frequency distribution of total scores using different combinations of values for C1 and C2 to visually identify the optimal fit for the left-skewed distribution expected in a healthy population.ResultsSerotype 1 had the highest prevalence of undetectable OI at baseline (77.0%, P < 0.001), and serotype 19A had the lowest (8.8%, P < 0.001). Immunogenicity was highest for serotype 7F (mean change of 18354, P < 0.001 for all comparisons). For vaccine response analysis, C1 = 300 and C2 = 8 produced a left-skewed distribution (Figure 2). Using these cutoffs, the median total score was 7 and the 5th percentile score was −1.ConclusionCriteria for normal MOPA results can be developed for single-timepoint data, or using a scoring system for vaccine response data that integrates pre-vaccine OI and fold-rise in OI. Additional studies in healthy and disease populations are needed to further optimize diagnostic criteria for discriminating normal vs. abnormal results. Disclosures All authors: No reported disclosures.

Characterization and Protective Activity of Monoclonal Antibodies Directed against Streptococcus suis Serotype 2 Capsular Polysaccharide Obtained Using a Glycoconjugate.

Streptococcus suis serotype 2 is an encapsulated bacterium and an important swine pathogen. Opsonizing antibody responses targeting capsular polysaccharides (CPSs) are protective against extracellular pathogens. To elucidate the protective activity of monoclonal antibodies (mAbs) directed against S. suis serotype 2 CPS, mice were immunized with a serotype 2 CPS-glycoconjugate and three hybridomas were isolated; of which, two were murine IgMs and the other a murine IgG1. Whereas the IgMs (mAbs 9E7 and 13C8) showed different reactivity levels with S. suis serotypes 1, 1/2, 2 and 14, the IgG1 (mAb 16H11) was shown to be serotype 2-specific. All mAbs targeted the sialylated chain of the CPSs. Using an opsonophagocytosis assay, the IgMs were opsonizing towards the S. suis serotypes to which they cross-react, while the IgG1 failed to induce bacterial elimination. In a model of mouse passive immunization followed by a lethal challenge with S. suis serotype 2, the IgG1 and IgM cross-reacting only with serotype 14 (mAb 13C8) failed to protect, while the IgM cross-reacting with serotypes 1, 1/2, and 14 (mAb 9E7) was shown to be protective by limiting bacteremia. These new mAbs show promise as new S. suis diagnostic tools, as well as potential for therapeutic applications.

A pathogen-free flow cytometry based opsonophagocytosis assay protocol to quantify antibody-mediated phagocytosis

Abstract An important function of antibodies is their ability to mark cells for phagocytosis and subsequent degradation in a process known as opsonization. When developing new vaccine candidates, it is critical to determine whether the antigen specific antibodies elicited in response to immunization have this function. Due to the high containment working constraints of several important human pathogens, it is not always possible to directly study this property using the pathogen of interest. Therefore, we sought to develop a pathogen-free, flow cytometry based assay to quantify the opsonizing activity of antibodies elicited in response to immunization. We studied this using vaccine candidates against Coxiella burnetii, the causative agent of Q-fever that is a potential bioterrorism agent. In our assay, fluorescent polystyrene beads were conjugated to C. burnetii proteins then incubated with the antibody-containing sera. The antibody-coated beads were incubated with macrophages and phagocytosis of the fluorescent beads was assayed by flow cytometry. This assay allows for rapid quantification of the opsonizing capacity of antigen specific antibodies elicited in response to immunization.

CbpM and CbpG of Streptococcus Pneumoniae Elicit a High Protection in Mice Challenged with a Serotype 19F Pneumococcus

Among many pneumococcal antigens, choline-binding proteins (CPBs) display a high immunogenicity in animal models. This study aims to determine the immunogenicity of CbpM, CbpG and CbpL proteins of Streptococcus pneumoniae in a mice model. The genes were cloned into pET21a expression vector and the recombinant proteins were produced. Mice were immunized with the purified recombinant proteins. Subsequently, the mice were challenged with S. pneumoniae ATCC 49619 (2×106 CFU) and their survival and bacterial clearances were followed 24 hours after infection. The antibody responses of the mice were determined by ELISA assay. The opsonophagocytosis assay was performed using rabbit's sera. Passive immunization was carried out using two doses of anti-CbPs antibodies. Finally, these mice were experimentally infected with virulent bacteria and the protective effects of two doses of 10 and 100 µg/mL by monitoring the survival rate and bacterial clearance were determined at 2, 3 and 7 days after bacterial challenge. The mice actively immunized with CbpM, CbpG and CbpL recombinant proteins showed survival rate of 100%, 85% and 75%, respectively. The survival rates among passively immunized mice groups which received 100 µg/mL dose of anti-CbpM, anti-CbpG and anti- CbpL were 50%, 50% and 25%, respectively. The rates of opsonization with rabbit's antibodies against CbpM, CbpG, and CbpL at 100 µg/mL doses was 45.6%, 14.7% and 82.3%, and at 10 µg/mL was 12.9%, 12.2% and 9.35%, respectively.Our findings suggest that the recombinant proteins particularly CbpM and CbpG can protect the mice against pneumococcus19F serotype and effectively induce a protective antibody response. Thus, CbpG and CbpM proteins might be used as suitable vaccine candidate in pneumococcal vaccine formulations.

Accurate prediction of anti-phagocytic activity of Vibrio vulnificus by measurement of bacterial adherence to hydrocarbonsPrediction of Anti-Phagocytic Activity.

Vibrio vulnificus can cause necrotizing soft tissue infection via exposure through an open wound, and the incubation period in cases of wound infection is only about 16h. These facts strongly suggest that mechanisms to evade innate immune cell phagocytosis are essential for its pathogenicity. Hydrophobic interaction is one of the binding mechanisms between bacteria and phagocytes. Several factors that maintain cell surface hydrophobicity (CSH) can contribute to anti-phagocytic activity. In this study, we tried to identify V.vulnificus genes involved in maintaining the CSH, in order to elucidate mechanisms of anti-phagocytic activity. We obtained 143 mutants that had lost their ability to proliferate in the host, using signature-tagged transposon basis mutagenesis (STM). The CSH of these mutants was measured by the bacterial adherence to hydrocarbons (BATH) assay. The CSH of only four mutants differed significantly from that of wild type (WT). Of these four mutants, degS mutant (degS::Tn) showed lesser anti-phagocytic activity than WT in the opsonophagocytosis assay, even though degS::Tn showed opaque-type colonies. Furthermore, survival times of mice subcutaneously inoculated with degS::Tn were prolonged. These facts indicated that the BATH assay is a more suitable method of analyzing the anti-phagocytic activity of V.vulnificus than the comparison of colony morphology.