scholarly journals Improvement of Immunogenicity of Meningococcal Lipooligosaccharide by Coformulation with Lipidated Transferrin-Binding Protein B in Liposomes: Implications for Vaccine Development

2012 ◽  
Vol 19 (5) ◽  
pp. 711-722 ◽  
Author(s):  
Noëlle Mistretta ◽  
Bruno Guy ◽  
Yves Bérard ◽  
François Dalençon ◽  
Olivia Fratantonio ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Binding Protein ◽  
Hek293 Cells ◽  
Molar Ratio ◽  
Adjuvant Effect ◽  
Content Type ◽  
Endotoxic Activity ◽  
Protein B

ABSTRACTAmong various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group BNeisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluatedin vitrousing theLimulusAmebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, anin vivostudy demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

scholarly journals The Combinations Chitosan-Pam3CSK4 and Chitosan-Monophosphoryl Lipid A: Promising Immune-Enhancing Adjuvants for Anticaries Vaccine PAc

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Yongli Bi ◽  
Qingan Xu ◽  
Lingkai Su ◽  
Jiantao Xu ◽  
Zhongfang Liu ◽  
...  
Keyword(s):  
Lipid A ◽  
Vaccine Development ◽  
Protection Effect ◽  
Content Type ◽  
Monophosphoryl Lipid A ◽  
Monophosphoryl Lipid ◽  
Tooth Surfaces ◽  
Antibody Titers

ABSTRACT We previously demonstrated that recombinant protein PAc could be administered as an anticaries vaccine. However, the relatively weak immunogenicity of PAc limits its application. In the present study, we investigated the effect of two adjuvant combinations of chitosan plus Pam3CSK4 (chitosan-Pam3CSK4) and of chitosan plus monophosphoryl lipid A (chitosan-MPL) in the immune responses to the PAc protein in vivo and in vitro. PAc-chitosan-Pam3CSK4 or PAc-chitosan-MPL promoted significantly higher PAc-specific antibody titers in serum and saliva, inhibited Streptococcus mutans colonization onto the tooth surfaces, and endowed better protection effect with significantly less caries activities than PAc alone. Chitosan-Pam3CSK4 and chitosan-MPL showed no statistically significant differences. In conclusion, our study demonstrated that the chitosan-Pam3CSK4 and chitosan-MPL combinations are promising for anticaries vaccine development.

scholarly journals Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

2012 ◽  
Vol 19 (10) ◽  
pp. 1603-1608 ◽  
Author(s):  
Koushik Roy ◽  
David J. Hamilton ◽  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Diarrheal Disease ◽  
Vaccine Development ◽  
Enterotoxigenic Escherichia Coli ◽  
Intestinal Colonization ◽  
Content Type ◽  
Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonizationin vivoand toxin delivery to epithelial cellsin vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development.

scholarly journals The Borrelia hermsii Factor H Binding Protein FhbA Is Not Required for Infectivity in Mice or for Resistance to Human ComplementIn Vitro

2014 ◽  
Vol 82 (8) ◽  
pp. 3324-3332 ◽  
Author(s):  
Lindy M. Fine ◽  
Daniel P. Miller ◽  
Katherine L. Mallory ◽  
Brittney K. Tegels ◽  
Christopher G. Earnhart ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Binding Protein ◽  
Factor H ◽  
Negative Regulator ◽  
Human Complement ◽  
Relapsing Fever ◽  
Borrelia Hermsii ◽  
Content Type

ABSTRACTThe primary causative agent of tick-borne relapsing fever in North America isBorrelia hermsii. It has been hypothesized thatB. hermsiievades complement-mediated destruction by binding factor H (FH), a host-derived negative regulator of complement.In vitro,B. hermsiiproduces a single FH binding protein designated FhbA (FH binding protein A). The properties and ligand binding activity of FhbA suggest that it plays multiple roles in pathogenesis. It binds plasminogen and has been identified as a significant target of a B1b B cell-mediated IgM response in mice. FhbA has also been explored as a potential diagnostic antigen forB. hermsiiinfection in humans. The ability to test the hypothesis that FhbA is a critical virulence factorin vivohas been hampered by the lack of well-developed systems for the genetic manipulation of the relapsing fever spirochetes. In this report, we have successfully generated aB. hermsiifhbAdeletion mutant (theB. hermsiiYORΔfhbAstrain) through allelic exchange mutagenesis. Deletion offhbAabolished FH binding by the YORΔfhbAstrain and eliminated cleavage of C3b on the cell surface. However, the YORΔfhbAstrain remained infectious in mice and retained resistance to killingin vitroby human complement. Collectively, these results indicate thatB. hermsiiemploys an FhbA/FH-independent mechanism of complement evasion that allows for resistance to killing by human complement and persistence in mice.

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

2018 ◽  
Vol 86 (7) ◽  
Author(s):  
Christopher R. Doyle ◽  
Jee-Young Moon ◽  
Johanna P. Daily ◽  
Tao Wang ◽  
Liise-anne Pirofski
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Vaccine Development ◽  
Lavage Fluid ◽  
Uptake System ◽  
Content Type

ABSTRACT 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.

scholarly journals Different Roles for Lactococcal Aggregation Factor and Mucin Binding Protein in Adhesion to Gastrointestinal Mucosa

2012 ◽  
Vol 78 (22) ◽  
pp. 7993-8000 ◽  
Author(s):  
Jovanka Lukić ◽  
Ivana Strahinić ◽  
Branko Jovčić ◽  
Brankica Filipić ◽  
Ljubiša Topisirović ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Hydrophobic Interactions ◽  
Binding Protein ◽  
Content Type ◽  
Mucosal Surfaces ◽  
Gastric Type ◽  
Aggregation Factor ◽  
Selection Of

ABSTRACTAdhesion of bacteria to mucosal surfaces and epithelial cells is one of the key features for the selection of probiotics. In this study, we assessed the adhesion property ofLactococcus lactissubsp.lactisBGKP1 based on its strong autoaggregation phenotype and the presence of the mucin binding protein (MbpL). Genes involved in aggregation (aggL) and possible interaction with mucin (mbpL), present on the same plasmid pKP1, were previously separately cloned in the plasmid pAZIL.In vivoandin vitroexperiments revealed potentially different physiological roles of these two proteins in the process of adherence to the intestine during the passage of the strain through the gastrointestinal tract. We correlated thein vitroandin vivoaggregation of the BGKP1-20 carrying plasmid withaggLto binding to the colonic mucus through nonspecific hydrophobic interactions. The expression of AggL on the bacterial cell surface significantly increased the hydrophobicity of the strain. On the other hand, the presence of AggL in the strain reduced its ability to adhere to the ileum. Moreover, MbpL protein showed an affinity to bind gastric type mucin proteins such as MUC5AC. This protein did not contribute to the binding of the strain to the ileal or colonic part of the intestine. Different potential functions of lactococcal AggL and MbpL proteins in the process of adhesion to the gastrointestinal tract are proposed.

scholarly journals Nonbinding Site-Directed Mutants of Transferrin Binding Protein B Exhibit Enhanced Immunogenicity and Protective Capabilities

2014 ◽  
Vol 83 (3) ◽  
pp. 1030-1038 ◽  
Author(s):  
Rafael Frandoloso ◽  
Sonia Martínez-Martínez ◽  
Charles Calmettes ◽  
Jamie Fegan ◽  
Estela Costa ◽  
...  
Keyword(s):  
Immune Response ◽  
Binding Protein ◽  
Iron Acquisition ◽  
Critical Role ◽  
T Cell Response ◽  
Content Type ◽  
Growth And Survival ◽  
Mutant Proteins ◽  
Protein B

Host-adapted Gram-negative bacterial pathogens from thePasteurellaceae,Neisseriaceae, andMoraxellaceaefamilies normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesisin vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutantHaemophilus parasuisTbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines.

scholarly journals Chemical Attenuation in the Development of a Whole-Organism Malaria Vaccine

2017 ◽  
Vol 85 (7) ◽  
Author(s):  
Amber I. Raja ◽  
Danielle I. Stanisic ◽  
Michael F. Good
Keyword(s):  
Subunit Vaccine ◽  
Malaria Parasite ◽  
Malaria Vaccine ◽  
Vaccine Development ◽  
Liver Stage ◽  
Content Type ◽  
Vaccine Candidates ◽  
Limited Efficacy

ABSTRACT Malaria vaccine development has been dominated by the subunit approach; however, many subunit vaccine candidates have had limited efficacy in settings of malaria endemicity. As our search for an efficacious malaria vaccine continues, the development of a whole-organism vaccine is now receiving much scrutiny. One strategy currently being explored in the development of a whole-organism vaccine involves chemical attenuation of the malaria parasite. In vivo and in vitro chemical attenuation of both liver-stage and blood-stage Plasmodium parasites has been investigated. Here, we discuss both approaches of chemical attenuation in the development of a whole-organism vaccine against malaria.

scholarly journals Adenylate Charge Regulates Sensor Kinase CheS3 To Control Cyst Formation in Rhodospirillum centenum

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Kuang He ◽  
Vladimira Dragnea ◽  
Carl E. Bauer
Keyword(s):  
Histidine Kinase ◽  
Developmental Process ◽  
Cyst Formation ◽  
Molar Ratio ◽  
Content Type ◽  
Rhodospirillum Centenum ◽  
Cellular Energy ◽  
Hybrid Histidine Kinase

ABSTRACTRhodospirillum centenumforms metabolically dormant cysts under unfavorable growth conditions such as desiccation or nutrient starvation. The development of cysts is tightly regulated and involves a cyst-repressing chemotaxis-like signal transduction pathway called the Che3signaling cascade. The Che3cascade is comprised of a methyl chemoreceptor (MCP3), receptor-methylating/demethylating proteins CheB3and CheR3, two CheW3linker proteins, a CheA3-CheY hybrid histidine kinase, and a single-domain response regulator, CheY3. In addition to Che-like components, the Che3cascade also contains a second hybrid histidine kinase, CheS3. Recent biochemical and genetic studies show that CheA3does not serve as a phosphor donor for CheY3; instead, CheA3inhibits a CheS3→CheY3two-component system by phosphorylating an inhibitory receiver domain of CheS3. In this study, we show that in addition to phosphorylation by CheA3, the phosphorylation state of CheS3is also regulated by the cellular energy level as quantified by the molar ratio of ATP/(ATP + ADP). A 35% decrease in cellular energy is shown to occurin vivoupon a nutrient downshift that gives rise to cyst formation. When this energy decline is replicatedin vitro, the phosphorylation level of CheS3is reduced by ~75%. Finally, we also show that ADP-mediated reduction of CheS3phosphorylation is a consequence of ADP enhancing autodephosphorylation of CheS3.IMPORTANCEUpon starvation,Rhodospirillum centenumundergoes a developmental process that forms metabolically dormant cysts, which withstand desiccation and nutritional limitation. This study explores the role of the cellular energy state as measured by the ratio of ATP to ADP as an important regulator of cyst formation inRhodospirillum centenum. We show thatR. centenumcells experience a significant reduction in ATP during cyst formation using ATP/(ATP + ADP) as a measurement. When thisin vivolevel of energy starvation is simulatedin vitro, CheS3phosphorylation is reduced by 75%. This profound reduction in CheS3autophosphorylation is contrasted with a much lower 25% decrease in CheA3phosphorylation in response to a similar downward shift in ATP/(ATP + ADP). We argue that even though adenylate energy affects all ATP-dependent enzymes to an extent, the enhanced inhibition of CheS3activity in response to a reduction in the ATP/(ATP + ADP) ratio likely functions as an important input signal to regulate cyst development.

scholarly journals Vitamin E Increases Antimicrobial Sensitivity by Inhibiting Bacterial Lipocalin Antibiotic Binding

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Marwa M. Naguib ◽  
Miguel A. Valvano
Keyword(s):  
Cystic Fibrosis ◽  
Vitamin E ◽  
Water Soluble ◽  
Burkholderia Cenocepacia ◽  
Bacterial Cells ◽  
Adjuvant Effect ◽  
Bacterial Killing ◽  
Content Type

ABSTRACT Burkholderia cenocepacia is an opportunistic Gram-negative bacterium that causes serious respiratory infections in patients with cystic fibrosis. Recently, we discovered that B. cenocepacia produces the extracellular bacterial lipocalin protein BcnA upon exposure to sublethal concentrations of bactericidal antibiotics. BcnA captures a range of antibiotics outside bacterial cells, providing a global extracellular mechanism of antimicrobial resistance. In this study, we investigated water-soluble and liposoluble forms of vitamin E as inhibitors of antibiotic binding by BcnA. Our results demonstrate that in vitro, both vitamin E forms bind strongly to BcnA and contribute to reduce the MICs of norfloxacin (a fluoroquinolone) and ceftazidime (a β-lactam), both of them used as model molecules representing two different chemical classes of antibiotics. Expression of BcnA was required for the adjuvant effect of vitamin E. These results were replicated in vivo using the Galleria mellonella larva infection model whereby vitamin E treatment, in combination with norfloxacin, significantly increased larva survival upon infection in a BcnA-dependent manner. Together, our data suggest that vitamin E can be used to increase killing by bactericidal antibiotics through interference with lipocalin binding. IMPORTANCE Bacteria exposed to stress mediated by sublethal antibiotic concentrations respond by adaptive mechanisms leading to an overall increase of antibiotic resistance. One of these mechanisms involves the release of bacterial proteins called lipocalins, which have the ability to sequester antibiotics in the extracellular space before they reach bacterial cells. We speculated that interfering with lipocalin-mediated antibiotic binding could enhance the efficacy of antibiotics to kill bacteria. In this work, we report that when combined with bactericidal antibiotics, vitamin E contributes to enhance bacterial killing both in vitro and in vivo. This adjuvant effect of vitamin E requires the presence of BcnA, a bacterial lipocalin produced by the cystic fibrosis pathogen Burkholderia cenocepacia. Since most bacteria produce lipocalins like BcnA, we propose that our findings could be translated into making novel antibiotic adjuvants to potentiate bacterial killing by existing antibiotics.

scholarly journals The FomA Porin from Fusobacterium nucleatum Is a Toll-Like Receptor 2 Agonist with Immune Adjuvant Activity

2012 ◽  
Vol 19 (7) ◽  
pp. 1093-1101 ◽  
Author(s):  
Deana N. Toussi ◽  
Xiuping Liu ◽  
Paola Massari
Keyword(s):  
Fusobacterium Nucleatum ◽  
Target Cells ◽  
Toll Like Receptor ◽  
Adjuvant Effect ◽  
Adjuvant Activity ◽  
Content Type ◽  
Enhanced Production ◽  
Immune Adjuvant

ABSTRACTMany bacterial components selectively activate immune and nonhematopoietic target cells via Toll-like receptor (TLR) signaling; modulation of such host responses defines the immune adjuvant properties of these bacterial products. For example, the outer membrane protein porins fromNeisseria,Salmonella, andShigellaare known TLR2 agonists with established systemic and mucosal immune adjuvanticity. Early work indicated that the FomA porin fromFusobacterium nucleatumhas immune adjuvant activity in mice. Using a purified recombinant FomA, we have verified its immune stimulatory properties and have defined a role for TLR2 signaling in itsin vitroandin vivoactivity. FomA induces interleukin 8 (IL-8) secretion and NF-κB-dependent luciferase activity in HEK cells expressing TLR2, IL-6 secretion, and cell surface upregulation of CD86 and major histocompatibility complex (MHC) II in primary B cells from wild-type mice, but it fails to activate cells from TLR2 knockout mice. Accordingly, the immune adjuvant activity of FomA is also TLR2 dependent. In a mouse model of immunization with ovalbumin (OVA), FomA induces enhanced production of OVA-specific IgM and IgG, including IgG1 and IgG2b antibodies, as well as enhanced secretion of IL-10 and IL-6, consistent with a Th2-type adjuvant effect. We also observe a moderate production of anti-FomA antibodies, suggesting that FomA is also immunogenic, a quality that is also TLR2 dependent. Therefore, modulation of host immune responses by FomA may be effective for targeting general host immunity not only to pathogens (as a novel TLR2 adjuvant) but also toF. nucleatumitself (as an antigen), expanding its use as a self-adjuvanted antigen in an immunization strategy against polymicrobial infections, including those byF. nucleatum.

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