Opsonic Antibodies Research Articles

A Capsular Polysaccharide-Specific Antibody Alters Streptococcus pneumoniae Gene Expression during Nasopharyngeal Colonization of Mice.

Pneumococcal conjugate vaccines (PCV) elicit opsonophagocytic (opsonic) antibodies to pneumococcal capsular polysaccharides (PPS) and reduce nasopharyngeal (NP) colonization by vaccine-included Streptococcus pneumoniae serotypes. However, nonopsonic antibodies may also be important for protection against pneumococcal disease. For example, 1E2, a mouse IgG1 monoclonal antibody (MAb) to the serotype 3 (ST3) PPS (PPS3), reduced ST3 NP colonization in mice and altered ST3 gene expression in vitro Here, we determined whether 1E2 affects ST3 gene expression in vivo during colonization of mice by performing RNA sequencing on NP lavage fluid from ST3-infected mice treated with 1E2, a control MAb, or phosphate-buffered saline. Compared to the results for the controls, 1E2 significantly altered the expression of over 50 genes. It increased the expression of the piuBCDA operon, which encodes an iron uptake system, and decreased the expression of dpr, which encodes a protein critical for resistance to oxidative stress. 1E2-mediated effects on ST3 in vivo required divalent binding, as Fab fragments did not reduce NP colonization or alter ST3 gene expression. In vitro, 1E2 induced dose-dependent ST3 growth arrest and altered piuB and dpr expression, whereas an opsonic PPS3 MAb, 5F6, did not. 1E2-treated bacteria were more sensitive to hydrogen peroxide and the iron-requiring antibiotic streptonigrin, suggesting that 1E2 may increase iron import and enhance sensitivity to oxidative stress. Finally, 1E2 also induced rapid capsule shedding in vitro, suggesting that this may initiate 1E2-induced changes in sensitivity to oxidative stress and gene expression. Our data reveal a novel mechanism of direct, antibody-mediated antibacterial activity that could inform new directions in antipneumococcal therapy and vaccine development.

Antibodies to Conserved Surface Polysaccharides Protect Mice Against Bacterial Conjunctivitis.

PurposeBacterial conjunctivitis is a major problem in ocular health. Little is known about protective immune effectors in the conjunctiva. We evaluated whether opsonic antibody to the conserved surface/capsular polysaccharide poly-N-acetyl glucosamine (PNAG) expressed by Streptococcus pneumoniae and Staphylococcus aureus was protective against bacterial conjunctivitis, as well as an antibody to the Pseudomonas aeruginosa surface polysaccharide alginate.MethodsBacteria were injected directly into the conjunctivae of either A/J mice or into conjunctivae of wild type C57Bl/6 mice for comparisons to responses of recombination activating gene 1-knock out (RAG 1 KO) or germ-free mice in the C57Bl/6 genetic background. Human IgG1 monoclonal antibodies (MAb) to either PNAG or alginate were administered as follows: direct injection of 10 μg into the conjunctivae or topical application onto the cornea 4, 24, and 32 hours post infection; or intraperitoneal injection of 200 μg 18 hours prior to and then 4, 24, and 32-hours postinfection. After 48 hours, eyes were scored for pathology, mice were euthanized, and CFU/conjunctiva was determined.ResultsAll methods of antibody administration reduced S. pneumoniae, S. aureus, or P. aeruginosa pathology and bacterial levels in the conjunctivae. Histopathologic analysis showed severe inflammatory cell infiltrates in conjunctivae of mice treated with control MAb, whereas immune mice showed only very mild cellular infiltration. The protective effect of MAb to PNAG was abolished in RAG 1 KO and germ-free mice.ConclusionsAntibodies to both PNAG and alginate demonstrated therapeutic efficacy in models of S. pneumoniae, S. aureus, and P. aeruginosa conjunctivitis, validating the protective capacity of antibodies to surface polysaccharides in distinct ocular tissues.

Immunization with Larrea divaricata Cav. proteins elicits opsonic antibodies against Pseudomonas aeruginosa and induces phagocytic activity of murine macrophages

Pseudomonas aeruginosa is an opportunistic pathogen implicated in nosocomial infections for which no vaccines have been approved. Larrea divaricata Cav. (Jarilla) is a widely spread plant in America and it is used in folk medicine to treat several pathologies. It has also been shown that antibodies elicited against Jarilla proteins of crude extract (JPCE) cross-react with proteins from gram-negative bacteria. In this study we aim to assess the contribution of anti-JPCE antibodies in the opsonophagocytosis of P. aeruginosa by murine macrophages. Levels of reactivity of anti-JPCE IgG and IgA antibodies against cell and membrane proteins suggest that these proteins induce a response that could favor opsonic bacterial recognition, which is important for the elimination of bacteria on mucous membranes, useful in the early stages of infection. Opsonophagocytosis assays also show that these antibodies could favor bacteria intake. These results together with previous observations that indicate that anti-JPCE antibodies are able to neutralize P. aeruginosa enzymes point L. divaricata proteins as candidates for vaccine development.

A Streptococcus pneumoniae Type 2 Oligosaccharide Glycoconjugate Elicits Opsonic Antibodies and Is Protective in an Animal Model of Invasive Pneumococcal Disease.

Invasive pneumococcal diseases (IPDs) remain the leading cause of vaccine-preventable childhood death, even though highly effective pneumococcal conjugate vaccines (PCVs) are used in national immunization programs in many developing countries. Licensed PCVs currently cover only 13 of the over 90 serotypes of Streptococcus pneumoniae (Sp), so nonvaccine serotypes are a major obstacle to the effective control of IPD. Sp serotype 2 (ST2) is such a nonvaccine serotype that is the main cause of IPD in many countries, including Nepal, Bangladesh, and Guatemala. Glycoconjugate vaccines based on synthetic oligosaccharides instead of isolated polysaccharides offer an attractive alternative to the traditional process for PCV development. To prevent the IPDs caused by ST2, we identified an effective ST2 neoglycoconjugate vaccine candidate that was identified using a medicinal chemistry approach. Glycan microarrays containing a series of synthetic glycans resembling portions of the ST2 capsular polysaccharide (CPS) repeating unit were used to screen human and rabbit sera and identify epitope hits. Synthetic hexasaccharide 2, resembling one repeating unit (RU) of ST2 CPS, emerged as a hit from the glycan array screens. Vaccination with neoglycoconjugates consisting of hexasaccharide 2 coupled to carrier protein CRM197 stimulates a T-cell-dependent B-cell response that induced CPS-specific opsonic antibodies in mice, resulting in killing of encapsulated bacteria by phagocytic activity. Subcutaneous immunization with neoglycoconjugate protected mice from transnasal challenge with the highly virulent ST2 strain NCTC 7466 by reducing the bacterial load in lung tissue and blood.

Immunoprotective potential of BamA, the outer membrane protein assembly factor, against MDR Acinetobacter baumannii

AbstarctAcinetobacter baumannii infections are responsible for major health problems in immunocompromised patients particularly in intensive care units. Due to rapid acquisition of and also inherent drug resistance, a vaccine is an effective treatment option against this pathogen. BamA, an outer membrane β-barrel assembly protein, was identified in A. baumannii as potential vaccine candidate by in silico analysis. The immunoprotective efficacy of this highly conserved protein was investigated against a virulent multidrug resistant clinical isolate using murine pneumonia model. Recombinant BamA elicited a high IgG antibody titer (160000) in mice. Opsonophagocytic killing assay showed non-neutrilizing, opsonizing antibodies with combinatorial bactericidal activity of antibodies and complement components. Active and passive immunization protected 80 and 60% mice respectively against intranasal challenge with lethal dose (109 CFU) of virulent A. baumannii along with efficient clearance of bacteria in mice lungs and reduction in levels of pro-inflammatory cytokines viz. TNF-α, IL-6 and IL-1β in sera and lung tissue homogenate. Increase in levels of IL-10, an anti-inflammatory cytokine and reduction of neutrophils in lungs facilitated the control of infection. This study demonstrates the potential of BamA as effective vaccine candidate and a promising target for antibody-based therapy to protect against MDR A. baumannii infections.

Dendrimeric peptides can confer protection against foot-and-mouth disease virus in cattle.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease in cloven-hoofed animals. A synthetic vaccine candidate consisting of dendrimeric peptides harbouring two copies of a B-epitope [VP1(136–154)] linked to a T-cell epitope [3A(21–35)] of FMDV confers protection to type O FMDV challenge in pigs. Herein we show in cattle that novel dendrimeric peptides bearing a T-cell epitope [VP1(21–40] and two or four copies of a B-cell epitope [VP1(135–160)] from type O1 Campos FMDV (termed B2T and B4T, respectively) elicited FMDV specific immune responses to similar levels to a commercial vaccine. Animals were challenged with FMDV and 100% of vaccinated cattle with B2T or B4T were protected to podal generalization. Moreover, bovines immunized with B4T were completely protected (with no clinical signs) against FMDV challenge after three vaccine doses, which was associated with titers of viral neutralizing antibodies in serum higher than those of B2T group (p< 0.05) and levels of opsonic antibodies similar to those of animals immunized with one dose of FMDV commercial vaccine. Bovines vaccinated with both dendrimeric peptides presented high levels of IgG1 anti FMDV in sera and in mucosa. When IgA in nasal secretions was measured, 20% or 40% of the animals in B2T or B4T groups respectively, showed anti-FMDV IgA titers. In addition, B2T and B4T peptides evoked similar consistent T cell responses, being recognized in vitro by lymphocytes from most of the immunized cattle in the proliferation assay, and from all animals in the IFN-γ production assay. Taken together, these results support the potential of dendrimers B2T or B4T in cattle as a highly valuable, cost-effective FMDV candidate vaccine with DIVA potential.

Construction, expression, purification and characterization of secretin domain of PilQ and triple PilA-related disulfide loop peptides fusion protein from Pseudomonas aeruginosa.

Objective(s):Infection with Pseudomonas aeruginosa has been a long-standing obstacle for clinical therapy due to the complexity of the genetics and pathogenesis, as well for widespread resistance to antibiotics, thus attaching great importance to explore effective vaccines for prevention and treatment. This paper focuses on the introduction of novel Pseudomonas aeruginosa type IV pili (T4P)-based fusion protein containing the secretin domain of PilQ and tandem PilA-related peptides.Materials and Methods:We surveyed the expression of the PilQ380-705-PilA fusion protein in-frame with pET26b vector in which a rigid linker was used between two polypeptides and flexible linkers were inserted between the three tandem repeats and each pilA domains. The transformants were expressed in Escherichia coli BL21. The reactivity of specific antisera to the fusion protein was assessed by ELISA. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis, and twitching inhibition assays.Results:The fusion protein was purified in high yield by osmotic shock method using HisTrap affinity column. The protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen is 1:128. Results of opsonophagocytosis assay revealed that the antibodies elevated to the fusion protein promoted phagocytosis of the PAO1 and 6266E strains, so that the twitching immobilization test confirmed these results.Conclusion:Due to excellent killing activity mediated by opsonic antibodies and efficient immobilization of the strains, it seems that PilQ380-705-PilA fusion protein could be a reliable candidate vaccine against P. aeruginosa infection.

Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms.

B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts.

Polymeric antigen BLSOmp31 in aluminium hydroxide induces serum bactericidal and opsonic antibodies against Brucella canis in dogs

Polymeric antigen BLSOmp31 is an immunogenic vaccine candidate that confers protection against Brucella canis in mice. In this preliminary study, the immunogenicity and safety of BLSOmp31 adsorbed to aluminum hydroxide gel (BLSOmp31-AH) were evaluated in Beagle dogs. In addition, the potential to elicit serum antibodies with complement-dependent bactericidal activity and/or to enhance phagocytosis by neutrophils were analyzed. Dogs were immunized three times with BLSOmp31-AH by subcutaneous route, followed by an annual booster. The vaccine elicited specific antibodies 3 weeks after the first immunization. Annual booster induced comparable antibody response as the primary series. Humoral immune response stimulated by BLSOmp31-AH did not interfere with routine agglutination test for canine brucellosis. Antibodies demonstrated a high complement-dependent bactericidal activity against B. canis. Moreover, opsonization by immune serum not only stimulated binding and uptake of the bacteria by neutrophils but effectively enhanced the destruction of B. canis. Specific IgG was detected in 3/4 immunized dogs in preputial secretions. The antibody profile corresponded to a marked Th2 response, since IgG1 prevailed over IgG2 and cellular immune response was not detected in vitro or in vivo. These results require further evaluation in larger field studies to establish the full prophylactic activity of BLSOmp31 against canine brucellosis.

CD47 Monoclonal Antibody SRF231 Is a Potent Inducer of Macrophage-Mediated Tumor Cell Phagocytosis and Reduces Tumor Burden in Murine Models of Hematologic Malignancies

The transmembrane protein CD47 is an immunoglobulin superfamily member involved in multiple cellular processes, including cell migration, adhesion and T cell function. The interaction between CD47 and signal regulatory protein alpha (SIRPα), an inhibitory protein expressed on macrophages, prevents phagocytosis of CD47-expressing cells. CD47 was originally identified as a tumor antigen on human ovarian cancer and was subsequently shown to be expressed on multiple human tumor types, including both hematologic and solid tumors. In many hematologic cancers, high CD47 expression is associated with poor clinical outcomes. CD47 is also expressed at low levels on virtually all non-malignant cells, and loss of expression or changes in membrane distribution may serve as markers of aged or damaged cells, particularly on red blood cells (RBC). High expression of CD47 on tumors blocks phagocytic uptake, subsequent antigen cross-presentation and T cell activation, which collectively contribute to tumor immune evasion. Agents that block the CD47-SIRPα interaction can restore phagocytic uptake of CD47+ target cells and lower the threshold for macrophage activation, which can enhance the efficacy of therapeutic antibodies with ADCC-enabling activity. We developed and characterized a CD47 blocking antibody and evaluated its activity in multiple hematologic models.SRF231 is a fully human monoclonal antibody that binds with high affinity to human CD47 and blocks the CD47-SIRP□ interaction. SRF231 promotes macrophage-mediated phagocytic clearance of several hematologic primary tumor samples and cell lines in vitro. For example, SRF231 increases phagocytosis of Raji tumor cell line targets with an EC50 of ~300 ng/ml. Enhanced phagocytosis is preferential for tumor cells over normal leukocytes and RBC. Tumor cell phagocytosis can be enhanced in the presence of opsonizing antibodies (e.g., anti-CD20 Ab) when co-administered with SRF231.In vivo efficacy of SRF231 was assessed in several preclinical murine xenograft models of hematologic malignancies. Notably, SRF231 administration led to profound tumor growth inhibition in models of multiple myeloma, diffuse large B cell lymphoma and Burkitt's lymphoma as a single agent and in combination with opsonizing antibodies. In the Raji xenograft model, single agent therapy leads to 112% tumor growth inhibition. This anti-tumor activity is at least partially dependent on macrophages, as depletion of macrophages via clodronate administration leads to reduced tumor growth inhibition. Tumor-associated macrophage (TAM) numbers and polarization status are also modulated by SRF231 treatment.In summary, the CD47 mAb SRF231 induces robust tumor cell phagocytosis and tumor clearance both alone and in combination with opsonizing antibodies in pre-clinical models of myeloma and lymphoma. These properties warrant further development of SRF231 in hematologic malignancies. SRF231 is currently in IND-enabling studies and is expected to enter clinical trials in 2017. DisclosuresNo relevant conflicts of interest to declare.

Association of serum bactericidal antibody and opsonic antibody levels after Neisseria meningitidis serogroup C conjugate vaccine in Brazilian children and adolescents infected or not infected with HIV

Neisseria meningitidis serogroup C (MenC) is the main causative agent of meningitis in Brazil. HIV infection affects the quality of the immune system. HIV+ children have an increased risk of infection to encapsulated bacteria such as N. meningitidis. We evaluated the opsonic antibody (OPA) levels and its correlation with serum bactericidal antibody (SBA) levels induced by one and two doses of a MenC conjugate vaccine in children and adolescents HIV+ and HIV-exposed but uninfected children (HEU) group. Overall the data show the importance of two doses of vaccine for HIV+ individuals. About 79% and 58% of HIV+ patients showed SBA and OPA positive response after two doses of vaccine, respectively. For HEU group, 62% and 41% of patients showed SBA and OPA positive response after one dose of vaccine, respectively. A positive and significant association between SBA and OPA levels was seen after two doses of vaccine in HIV+ patients.

Immunization with Protein D from Non-Typeable Haemophilus influenzae (NTHi) Induced Cytokine Responses and Bioactive Antibody Production.

BackgroundOuter membrane protein D (PD) is a highly conserved and stable protein in the outer membrane of both encapsulated (typeable) and non-capsulated (non-typeable) strains of Haemophilus influenzae. As an immunogen, PD is a potential candidate vaccine against non-typeable H. influenzae (NTHi) strains.ObjectivesThe aim of this study was to determine the cytokine pattern and the opsonic antibody response in a BALB/c mouse model versus PD from NTHi as a vaccine candidate.MethodsProtein D was formulated with Freund’s and outer membrane vesicle (OMV) adjuvants and injected into experimental mice. Sera from all groups were collected. The bioactivity of the anti-PD antibody was determined by opsonophagocytic killing test. To evaluate the cytokine responses, the spleens were assembled, suspension of splenocytes was recalled with antigen, and culture supernatants were analyzed by ELISA for IL-4, IL-10, and IFN-γ cytokines.ResultsAnti-PD antibodies promoted phagocytosis of NTHi in both immunized mice groups (those administered PD + Freund’s and those administered PD + OMV adjuvants, 92.8% and 83.5%, respectively, compared to the control group). In addition, the concentrations of three cytokines were increased markedly in immunized mice.ConclusionsWe conclude that immunization with PD protects mice against NTHi. It is associated with improvements in both cellular and humoral immune responses and opsonic antibody activity.

Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies.

This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4+ T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value.

BscF from Bordetella pertussis provides advantageous adjuvant activity when paired with the acellular pertussis vaccine: enhanced Th1/Th17 pertussis-specific immune response

Abstract Incidence of pertussis, a severe respiratory disease caused by Bordetella pertussis, have been on the rise. This resurgence has been linked to antigenic divergence in circulating pertussis strains as well as waning or ineffective immunity induced by the current acellular pertussis (aP) vaccine. The current aP vaccine replaced a whole cell pertussis (wP) vaccine in the 1990s due to adverse events associated with the wP vaccine. Alum-absorbed aP vaccine has been shown to elicit a strong antibody response and considerable Th2 type CD4+ T cells. In contrast, wP vaccine promotes Th1/Th17 type cellular immunity and associated opsonizing antibodies presumably via its PAMPs. We have previously demonstrated that BscF, a B. pertussis needle protein, acts as a strong TLR agonist. Here we characterized the contribution of BscF to a laboratory prepared aP vaccine-induced response. aP + BscF resulted in statistically greater pertussis-specific antibody titers, compared to sham aP + PBS. BscF enhanced long-term aP-specific immunity, compared to either the alum or sham controls, as measured by increased numbers of central memory T cells. Moreover, the addition of BscF resulted in skewing the pertussis-specific immunity toward Th1 and Th17 responses, compared to the addition of alum or PBS which skewed pertussis-specific immunity away from Th1/Th17 responses. Mice immunized with aP + BscF demonstrated significantly reduced bacterial burden in their lungs 5 dpi. These findings suggest that BscF induced a strong Th1 and Th17 anti-pertussis response, potentially may provide protective BscF-specific immunity, and could be a novel additional component in the next generation aP vaccine.

Unveiling Unexpected Immune Activities Induced by Your Pneumococcal Vaccine.

ABSTRACTIn modern-day vaccine design, a good pneumococcal capsular polysaccharide vaccine is measured by its ability to induce opsonic antibodies. These antibodies label bacteria for phagocytosis by neutrophils and thereby overcome the capsule’s barrier function. Doyle and Pirofski have raised a serious challenge to the current paradigm by describing anti-capsular antibodies that are highly protective but nonopsonic [C.R. Doyle and L. Pirofski, mBio 7(1):e02260-15, 2016, doi:10.1128/mBio.02260-15]. In fact, some functions are not related to neutrophils or phagocytosis at all. An increased awareness of these activities is critical not only for accurate comparisons of vaccine candidates but also for improvements in vaccination outcomes in settings of neutropenia. When vaccine developers select a single gatekeeper assay (e.g., an opsonophagocytic assay for bacteria or a neutralization assay for viruses), promising vaccine candidates may be missed. Doyle and Pirofski stress that multiple functions, not just one, should be investigated to enhance discovery of antibody mechanisms and to best assess vaccine-induced correlates of immune protection.

Reduction of Streptococcus pneumoniae Colonization and Dissemination by a Nonopsonic Capsular Polysaccharide Antibody.

ABSTRACTStreptococcus pneumoniae colonization of the nasopharynx (NP) is a prerequisite for invasive pneumococcal disease (IPD). The marked reduction in IPD that followed the routine use of pneumococcal polysaccharide conjugate vaccines (PCVs) has been linked to reduced NP colonization with vaccine-included serotypes (STs), with the caveat that PCVs are less effective against pneumonia than against IPD. Although PCV-elicited opsonic antibodies that enhance phagocytic killing of the homologous ST are considered a key correlate of PCV-mediated protection, recent studies question this relationship for some STs, including ST3. Studies with monoclonal antibodies (MAbs) to the pneumococcal capsular polysaccharide (PPS) of ST3 (PPS3) have shown that nonopsonic, as well as opsonic, antibodies can each protect mice against pneumonia and sepsis, but the effect of these types of MAbs on NP colonization is unknown. In this study, we determined the effects of protective opsonic and nonopsonic PPS3 MAbs on ST3 NP colonization in mice. Our results show that a nonopsonic MAb reduced early NP colonization and prevented ST3 dissemination to the lungs and blood, but an opsonic MAb did not. Moreover, the opsonic MAb induced a proinflammatory NP cytokine response, but the nonopsonic MAb had an antiinflammatory effect. The effect of the nonopsonic MAb on colonization did not require its Fc region, but its antiinflammatory effect did. Our findings challenge the paradigm that opsonic MAbs are required to prevent NP colonization and suggest that further studies of the activity of nonopsonic antibodies could advance our understanding of mechanisms of PCV efficacy and provide novel correlates of protection.

Characterization of Two Metal Binding Lipoproteins as Vaccine Candidates for Enterococcal Infections.

Background Enterococcus faecium and faecalis are Gram-positive opportunistic pathogens that have become leading causes of nosocomial infections over the last decades. Especially multidrug resistant enterococci have become a challenging clinical problem worldwide. Therefore, new treatment options are needed and the identification of alternative targets for vaccine development has emerged as a feasible alternative to fight the infections caused by these pathogens.ResultsWe extrapolate the transcriptomic data from a mice peritonitis infection model in E. faecalis to identify putative up-regulated surface proteins under infection conditions in E. faecium. After the bionformatic analyses two metal binding lipoproteins were identified to have a high homology (>72%) between the two species, the manganese ABC transporter substrate-binding lipoprotein (PsaAfm,) and the zinc ABC transporter substrate-binding lipoprotein (AdcAfm). These candidate lipoproteins were overexpressed in Escherichia coli and purified. The recombinant proteins were used to produce rabbit polyclonal antibodies that were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Mice were passively immunized with the antibodies raised against recombinant lipoproteins, showing significant reduction of colony counts in mice livers after the bacterial challenge and demonstrating the efficacy of these metal binding lipoproteins as promising vaccine candidates to treat infections caused by these enterococcal pathogens.ConclusionOverall, our results demonstrate that these two metal binding lipoproteins elicited specific, opsonic and protective antibodies, with an extensive cross-reactivity and serotype-independent coverage among these two important nocosomial pathogens. Pointing these two protein antigens as promising immunogens, that can be used as single components or as carrier proteins together with polysaccharide antigens in vaccine development against enterococcal infections.

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

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

ITOC2 – 031. Immunomodulatory characteristics of Resminostat, a novel HDAC inhibitor in phase II clinical development

Background Anti-cancer effects of HDAC inhibitors were shown to rely on an intact immune system. Additionally, combination of HDAC inhibitors with different cancer immunotherapy approaches was shown to be successful, suggesting that HDAC inhibition itself may engage and mediate an anti-tumoural immune response during cancer therapy. Aim Evaluation of immunomodulating effects of the HDAC inhibitor Resminostat, potentially contributing to its anti-tumoural activity. Method Resminostat effects on mechanisms affecting anti-tumoural immune responses were analysed in hepatocellular HepG2, Huh7, and SNU475 and adenocarcinomic A549 cells. Results Resminostat strongly reduced the expression of immunosuppression mediating enzymes, IDO1 and ARG1. Additionally, Resminostat considerably enhanced the expression of NKG2D ligands, MHC class I molecules, and several cancer testis antigens on several tumour cell lines. Interestingly, Resminostat was also able to up-regulate the expression of MHC class II and co-stimulatory molecule 4-1BBL. Discussion&conclusions Resminostat showed promising immunomodulatory effects demonstrating potential synergism with immunotherapy approaches using opsonizing antibodies (e.g. rituximab), immunostimulating agents (e.g. cytokines, stimulating antibodies, TLR ligands), and immune checkpoint blockers (PD1/PDL1 and CTLA-4 inhibitors).

Parenteral immunization with polymeric subcellular vaccine BLSOmp31 induces serum bactericidal and opsonic antibodies against Brucella canis, and stimulates a local immune response in dogs

Parenteral immunization with polymeric subcellular vaccine BLSOmp31 induces serum bactericidal and opsonic antibodies against Brucella canis, and stimulates a local immune response in dogs