Utility of Hybrid Transferrin Binding Protein Antigens for Protection Against Pathogenic Neisseria Species

The surface transferrin receptor proteins from Neisseria gonorrhoeae have been recognized as ideal vaccine targets due to their critical role in survival in the human male genitourinary tract. Recombinant forms of the surface lipoprotein component of the receptor, transferrin binding protein B (TbpB), can be readily produced at high levels in the Escherichia coli cytoplasm and is suitable for commercial vaccine production. In contrast, the integral outer membrane protein, transferrin binding protein A (TbpA), is produced at relatively low levels in the outer membrane and requires detergents for solubilization and stabilization, processes not favorable for commercial applications. Capitalizing on the core β-barrel structural feature common to the lipoprotein and integral outer membrane protein we engineered the lipoprotein as a scaffold for displaying conserved surface epitopes from TbpA. A stable version of the C-terminal domain of TbpB was prepared by replacing four larger exposed variable loops with short linking peptide regions. Four surface regions from the plug and barrel domains of Neisseria TbpA were transplanted onto this TbpB C-lobe scaffold, generating stable hybrid antigens. Antisera generated in mice and rabbits against the hybrid antigens recognized TbpA at the surface of Neisseria meningitidis and inhibited transferrin-dependent growth at levels comparable or better than antisera directed against the native TbpA protein. Two of the engineered hybrid antigens each elicited a TbpA-specific bactericidal antibody response comparable to that induced by TbpA. A hybrid antigen generated using a foreign scaffold (TbpB from the pig pathogen Haemophilus parasuis) displaying neisserial TbpA loop 10 was evaluated in a model of lower genital tract colonization by N. gonorrhoeae and a model of invasive infection by N. meningitidis. The loop 10 hybrid antigen was as effective as full length TbpA in eliminating N. gonorrhoeae from the lower genital tract of female mice and was protective against the low dose invasive infection by N. meningitidis. These results demonstrate that TbpB or its derivatives can serve as an effective scaffold for displaying surface epitopes of integral outer membrane antigens and these antigens can elicit protection against bacterial challenge

Developing a PmSLP3-based vaccine formulation that provides robust long-lasting protection against hemorrhagic septicemia–causing serogroup B and E strains of Pasteurella multocida in cattle

BackgroundPasteurella multocida is a bacterial pathogen that causes a variety of infections across diverse animal species, with one of the most devastating associated diseases being hemorrhagic septicemia. Outbreaks of hemorrhagic septicemia in cattle and buffaloes are marked by rapid progression and high mortality. These infections have particularly harmful socio-economic impacts on small holder farmers in Africa and Asia who are heavily reliant on a small number of animals kept as a means of subsistence for milk and draft power purposes. A novel vaccine target, PmSLP-3, has been identified on the surface of hemorrhagic septicemia–associated strains of P. multocida and was previously shown to elicit robust protection in cattle against lethal challenge with a serogroup B strain.MethodsHere, we further investigate the protective efficacy of this surface lipoprotein, including evaluating the immunogenicity and protection upon formulation with a variety of adjuvants in both mice and cattle.ResultsPmSLP-3 formulated with Montanide ISA 61 elicited the highest level of serum and mucosal IgG, elicited long-lasting serum antibodies, and was fully protective against serogroup B challenge. Studies were then performed to identify the minimum number of doses required and the needed protein quantity to maintain protection. Duration studies were performed in cattle, demonstrating sustained serum IgG titres for 3 years after two doses of vaccine and full protection against lethal serogroup B challenge at 7 months after a single vaccine dose. Finally, a serogroup E challenge study was performed, demonstrating that PmSLP-3 vaccine can provide protection against challenge by the two serogroups responsible for hemorrhagic septicemia.ConclusionTogether, these data indicate that PmSLP-3 formulated with Montanide ISA 61 is an immunogenic and protective vaccine against hemorrhagic septicemia-causing P. multocida strains in cattle

Murine host response to Neisseria gonorrhoeae upper genital tract infection reveals a common transcriptional signature, plus distinct inflammatory responses that vary between reproductive cycle phases

Abstract Background The emergence of fully antimicrobial resistant Neisseria gonorrhoeae has led global public health agencies to identify a critical need for next generation anti-gonococcal pharmaceuticals. The development and success of these compounds will rely upon valid pre-clinical models of gonorrhoeae infection. We recently developed and reported the first model of upper genital tract gonococcal infection. During initial characterization, we observed significant reproductive cycle-based variation in infection outcome. When uterine infection occurred in the diestrus phase, there was significantly greater pathology than during estrus phase. The aim of this study was to evaluate transcriptional profiles of infected uterine tissue from mice in either estrus or diestrus phase in order to elucidate possible mechanisms for these differences. Results Genes and biological pathways with phase-independent induction during infection showed a chemokine dominant cytokine response to Neisseria gonorrhoeae. Despite general induction being phase-independent, this common anti-gonococcal response demonstrated greater induction during diestrus phase infection. Greater activity of granulocyte adhesion and diapedesis regulators during diestrus infection, particularly in chemokines and diapedesis regulators, was also shown. In addition to a greater induction of the common anti-gonococcal response, Gene Set Enrichment Analysis identified a diestrus-specific induction of type-1 interferon signaling pathways. Conclusions This transcriptional analysis of murine uterine gonococcal infection during distinct points in the natural reproductive cycle provided evidence for a common anti-gonococcal response characterized by significant induction of granulocyte chemokine expression and high proinflammatory mediators. The basic biology of this host response to N. gonorrhoeae in estrus and diestrus is similar at the pathway level but varies drastically in magnitude. Overlaying this, we observed type-1 interferon induction specifically in diestrus infection where greater pathology is observed. This supports recent work suggesting this pathway has a significant, possibly host-detrimental, function in gonococcal infection. Together these findings lay the groundwork for further examination of the role of interferons in gonococcal infection. Additionally, this work enables the implementation of the diestrus uterine infection model using the newly characterized host response as a marker of pathology and its prevention as a correlate of candidate vaccine efficacy and ability to protect against the devastating consequences of N. gonorrhoeae-associated sequelae

Global Analysis of Neutrophil Responses to <i>Neisseria gonorrhoeae</i> Reveals a Self-Propagating Inflammatory Program

<div><p>An overwhelming neutrophil-driven response causes both acute symptoms and the lasting sequelae that result from infection with <i>Neisseria gonorrhoeae</i>. Neutrophils undergo an aggressive opsonin-independent response to <i>N. gonorrhoeae</i>, driven by the innate decoy receptor CEACAM3. CEACAM3 is exclusively expressed by human neutrophils, and drives a potent binding, phagocytic engulfment and oxidative killing of Opa-expressing bacteria. In this study, we sought to explore the contribution of neutrophils to the pathogenic inflammatory process that typifies gonorrhea. Genome-wide microarray and biochemical profiling of gonococcal-infected neutrophils revealed that CEACAM3 engagement triggers a Syk-, PKCδ- and Tak1-dependent signaling cascade that results in the activation of an NF-κB-dependent transcriptional response, with consequent production of pro-inflammatory cytokines. Using an <i>in vivo</i> model of <i>N. gonorrhoeae</i> infection, we show that human CEACAM-expressing neutrophils have heightened migration toward the site of the infection where they may be further activated upon Opa-dependent binding. Together, this study establishes that the role of CEACAM3 is not restricted to the direct opsonin-independent killing by neutrophils, since it also drives the vigorous inflammatory response that typifies gonorrhea. By carrying the potential to mobilize increasing numbers of neutrophils, CEACAM3 thereby represents the tipping point between protective and pathogenic outcomes of <i>N. gonorrhoeae</i> infection.</p></div

<i>N. gonorrhoeae</i> infection elicits an NF-κB and p38 MAPK signaling-dependent cytokine response in CEABAC neutrophils.

<p>CEABAC-Opa interaction leads to activation of NF-κB signaling. (<b>A</b>) WT and CEABAC PMNs were left untreated or pre-incubated with DPI (10 µM) or cytochalasin D (10 mg/ml). PMNs were then infected with either Opa⁻ or Opa⁺<i>N. gonorrhoeae</i> and MIP-1α levels were measured 3 h post infection. N = 3, error bars represent SEM. One-Way ANOVA indicates no significant differences between corresponding samples. (<b>B</b>) WT and CEABAC PMNs were infected with Opa⁺<i>N. gonorrhoeae</i> (MOI = 10 bacteria/PMN) for times indicated, and levels of IkBα were then detected by immunoblot with IκBα antibody. Assessment of tubulin levels confirmed equal protein loading. (<b>C, D</b>) CEABAC-Opa interaction promotes activation of p38 MAPK. WT and CEABAC PMNS were infected as in (<b>B</b>). Activation of MAP kinases p38, Erk1, and Erk2 was determined by immunoblot with phospho-specific antibodies. Total p38 and Erk1/2 levels were assessed to ensure equal protein loading. (<b>D, F</b>) WT and CEABAC PMNs were left untreated or preincubated with (<b>D</b>) p38 inhibitor (SB203580, 10 µM) or (<b>E</b>) TAK1 inhibitor ((5z)-7-oxozeaenol, 500 nM). PMNs were then infected with either Opa⁻ or Opa⁺<i>N. gonorrhoeae</i> and MIP-1α and MIP-2 levels were measured 3 h post infection. N≥3, error bars represent SEM. One-Way ANOVA (with Tukey's post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001. Stars indicate significance against all other conditions.</p

CEACAM binding stimulates the inflammatory response to <i>N. gonorrhoeae in vivo</i>.

<p>(<b>A</b>) Neutrophil migration assay. PMN migration speed towards <i>N. gonorrhoeae</i> infected CEABAC or WT neutrophil-derived supernatants was measured using a Zigmond chamber. One-Way ANOVA (with Tukey's post-test) was performed for relevant samples, ***P<0.001 (<b>B</b>) Neutrophil infiltration is more pronounced in human CEACAM-expressing mice in an infected subcutaneous air pouch model. Manual neutrophil counts of wash fluids collected from air pouches. ‘PBS’ denotes mice injected with sterile PBS. One-Way ANOVA was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001 (<b>C</b>) Giemsa-Wright stain of wash fluid collected from CEABAC air pouch infected with Opa⁺<i>N. gonorrhoeae</i>. The arrows point towards <i>N. gonorrhoeae</i> inside the neutrophil. (<b>D</b>) Neutrophil-expressed CEACAMs mediate <i>N. gonorrhoeae</i> binding within the air pouch. Cells from trypsinized air pouches were grown on coverslips in the presence of antibiotics, and infected <i>in vitro</i> with Opa⁺<i>N. gonorrhoeae</i>. Cells were visualized by staining for filamentous actin <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004341#ppat.1004341-Lee1" target="_blank">[16]</a>, CEACAMs (green), DNA (blue) and bacteria (cyan). (<b>E</b>) Levels of MIP-1α, MIP-2, KC, and IL-1β were measured in wash fluids collected from air pouches. PMN⁻ refers to mice in which neutrophils were depleted by administration of the Gr1-specific clone RB6-8C5 antibody one day prior to infection with Opa⁺<i>N. gonorrhoeae</i>. One-Way ANOVA (with Tukey's post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001 (<b>F</b>) Inhibition of pro-inflammatory signaling reduces neutrophil infiltration into the air pouch. Neutrophil counts of wash fluids collected from CEABAC mice infected with Opa⁺<i>N. gonorrhoeae</i> in the presence or absence of TAK1 inhibitor.</p

Opa-expressing <i>N. gonorrhoeae</i> drive CEACAM-dependent production of pro-inflammatory cytokines in neutrophils.

<p>(<b>A–B</b>) CEABAC PMNs secret pro-inflammatory cytokines in response Opa⁺<i>N. gonorrhoeae</i>. Mouse (WT and CEABAC) neutrophils were infected with either Opa⁻ or Opa⁺<i>N. gonorrhoeae</i> (MOI 10). MIP-1α, MIP-2, KC and TNFα production was measured 3 h post infection at (<b>A</b>) protein and (<b>B</b>) mRNA level. N≥3, error bars represent SEM. (<b>C</b>) Human neutrophils infected with Opa⁺<i>N. gonorrhoeae</i> show increased levels of MIP-1α, MIP-2, TNFα, and IL-1α mRNA relative to PMNs infected with Opa⁻ bacteria. Each symbol represents an individual donor. Cytokine mRNA levels shown as relative to levels of GAPDH mRNA. One-Way ANOVA (with Tukey's post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001. For (<b>A</b>) and (<b>B</b>) stars indicate significance against all other conditions.</p

<i>N. gonorrhoeae</i> drives an acute inflammatory transcriptional program.

<p>A gene array of WT and CEABAC PMNs infected with Opa⁺<i>N. gonorrhoeae</i> was performed, and genes showing a significant increase in transcription over uninfected controls are illustrated. Genes with ≥2 fold induction over uninfected controls in both WT and CEABAC neutrophils 1 h post-infection are shown. (<b>A</b>) List of genes up-regulated to a similar level in both WT and CEABAC neutrophils relative to uninfected neutrophils. (<b>B</b>) List of genes differentially expressed between CEABAC and WT PMNs. The fold difference by which WT and CEABAC differ is indicated next to each bar.</p

CEACAM3 signaling is required for the PMN cytokine response to <i>N. gonorrhoeae</i>.

<p>Inhibition of Src-family kinases and Syk leads to decreased cytokine production by infected PMNs. (<b>A, B</b>) WT and CEABAC PMNs were left untreated or pre-incubated with (<b>A</b>) Src-family kinase inhibitor (PP2, 10 µM), or (<b>B</b>) Syk inhibitor (piceatannol, 50 µM). PMNs were then infected with either non-opaque (Opa⁻) or Opa-expressing (Opa⁺) <i>N. gonorrhoeae</i>, and MIP-1α and MIP-2 levels were measured 3 h post infection. N≥3, error bars represent SEM. One-Way ANOVA was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001 (<b>C</b>) Opa⁺<i>N. gonorrhoeae</i> infection leads to phosphorylation of PKCδ. WT and CEABAC PMNs were infected with Opa⁺<i>N. gonorrhoeae</i> (MOI = 10 bacteria/PMN) for times indicated times. PKCδ activation was measured by immunoblot using phospho-PKCδ antibody. Immunoblot for PKCδ indicates equal protein loading. (<b>D</b>) CEABAC PMNs were left untreated or pre-incubated with PKC inhibitor (BIS II, 10 µM). PMNs were then infected with Opa⁻ or Opa⁺<i>N. gonorrhoeae</i>, and MIP-1α levels were measured 3 h post infection. One-Way ANOVA (with Tukey's post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001. Unless otherwise indicated, stars indicate significance against all other conditions. (<b>E, F</b>) Inhibition of SFK, Syk, TAK1 or p38 does not affect bacterial binding (<b>E</b>) or phagocytosis (<b>F</b>). WT and CEABAC PMNs were left untreated or pre-incubated with indicated inhibitors. PMNs were then infected with Opa⁺<i>N. gonorrhoeae</i> (MOI = 25) for 30 min. Intracellular and total bacteria were differentially stained, and quantified via immunofluorescence microscopy. (<b>G</b>) Schematic representation of proposed signaling pathway resulting in bacterial engulfment, activation of oxidative burst and degranulation, and cytokine production.</p

Human CEACAM expression by mouse neutrophils results in neisserial capture and internalization.

<p>(<b>A</b>) Human CEACAMs are expressed in CEABAC neutrophils in a manner reflecting that in human neutrophils. Human neutrophils (top), or wild type (WT) and CEABAC mouse neutrophils (bottom) were fixed and stained with antibodies specific for CEACAM1, CEACAM3, CEACAM6, or a mouse IgG isotype control, and analyzed by flow cytometry. Isotype histograms are shaded. (<b>B</b>) Immunoblot showing CEACAM expression in WT and CEABAC neutrophils. (<b>C</b>) Mouse neutrophils do not bind <i>N. gonorrhoeae</i>, while human neutrophils bind <i>N. gonorrhoeae</i> in an Opa-dependent manner. Mouse (top) or human (bottom) neutrophils were infected with non-opaque (Opa⁻) or Opa-expressing (Opa⁺) <i>N. gonorrhoeae</i>. Cells were visualized by staining filamentous actin with phalloidin <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004341#ppat.1004341-Lee1" target="_blank">[16]</a>, and bacteria are shown in green. Intracellular and total bacteria were differentially stained, and quantified via immunofluorescence microscopy (<b>D</b>). (<b>E</b>) WT and CEABAC PMNs kill Opa⁻ and Opa⁺ bacteria with similar kinetics. Adherent WT and CEABAC PMNs were infected with either Opa⁻ or Opa⁺<i>N. gonorrhoeae</i> at an MOI = 1. Bacterial survival over time was evaluated as CFUs present in PMN lysates at each time point relative to bacterial CFUs present at time 0. N = 2. (<b>F–G</b>) <i>N. gonorrhoeae</i> infected CEABAC neutrophils respond analogously to human PMNs. Human (<b>F</b>) or mouse (WT and CEABAC) (<b>G</b>) neutrophils were infected with Opa⁻ or Opa⁺<i>N. gonorrhoeae</i> and oxidative burst and degranulation responses were analyzed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004341#s4" target="_blank">Materials and Methods</a>.</p