Acellular pertussis vaccine toxoided with carbodiimide

Pertussis toxin (PT) and filamentous haemagglutinin (FHa) were extracted from the culture fluid of Bo pertussis grown in (1) modified Stainer and Scholte liquid medium (SS-X), static, (2) SS-X medium plus cyclodextrin, shaken, (3) eyelodextrin-liquid (CL) medium, shaken, by a single-step procedure using dye-ligand affinity chromatography. The addition of cyclodextrin to SS-X medium and aeration greatly enhanced the amount of PT and FHA extracted with Blue Sepharose, and the resulting PT and FHA ratio in the antigen preparation depended on the cultural conditions. With cyclodextrin-supplemented media (SS-X, CL) and aeration, it appeared that the total protein extracted was accounted for as PT and FHA, whereas with SS-X (static), only approximately 507o (w/w) was accounted for as both antigens. Culture of B. pertussis in SS-X medium supplemented with cyclodextrin (shaken), yielded the most PT, whereas growth in the CL medium (shaken) yielded the most FHa, Minor protein components were detected in many of the PT-FHa preparations, and endotoxin was present to 1-27, (w/w) of total protein in antigen preparations AP-16 and AP-17. The mixture of PT and FHa was toxoided with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, HCl (EDAC), The toxoiding conditions most suitable for elimination of the pathophysiological activities of the A-protomer and B-oligomer of PT were reaction of EDAC with protein at a ratio of 80:1 by weight, at 3

Neisseria gonorrhoeae challenge increases matrix metalloproteinase-8 expression in fallopian tube explants

Indexación: Scopus.Background: Neisseria gonorrhoeae (Ngo) is the etiological agent of gonorrhea, a sexually transmitted infection that initially infects the female lower genital tract. In untreated women, the bacteria can ascend to the upper genital reproductive tract and infect the fallopian tube (FTs), which is associated with salpingitis and can lead to impaired FT function and infertility. The extracellular matrix (ECM) plays an important role in cell migration and differentiation in the female genital tract, and some pathogens modify the ECM to establish successful infections. The ECM is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), their endogenous inhibitors; MMP deregulation causes pathological conditions in a variety of tissues. Results: The aim of this work was to analyze the expression and localization of MMP-3, MMP-8, MMP-9, and TIMP-1 in FT explants during Ngo infection using real-time PCR, immunohistochemistry, zymography and ELISA. No significant variations in MMP-3, MMP-9, and TIMP-1 transcript levels were observed. In contrast, a significant increase (p < 0.05) was observed for MMP-8 expression and was accompanied by stromal immunoreactivity in infected explants. ELISA results supported these findings and showed that MMP-8 release increased upon gonococcal infection. Conclusions: Our results indicate that gonococcal infection induces increased MMP-8 expression, which might contribute to FT damage during infection. © 2017 Juica, Rodas, Solar, Borda, Vargas, Muñoz, Paredes, Christodoulides and Velasquez.https://www.frontiersin.org/articles/10.3389/fcimb.2017.00399/ful

Vaccine potential and diversity of the putative cell binding factor (CBF, NMB0345/NEIS1825) protein of Neisseria meningitidis

The cbf gene from Neisseria meningitidis strain MC58 encoding the putative Cell Binding Factor (CBF, NMB0345/NEIS1825) protein was cloned into the pRSETA system and a ~36-kDa recombinant (r)CBF protein expressed in Escherichia coli and purified by metal affinity chromatography. High titres of rCBF antibodies were induced in mice following immunization with rCBF-saline, rCBF-Al(OH)3, rCBF-Liposomes or rCBF-Zwittergent (Zw) 3–14 micelles, both with and without incorporated monophosphoryl lipid A (MPLA) adjuvant. Anti-rCBF sera reacted in western blots of meningococcal lysates with a single protein band of molecular mass ~29.5 kDa, indicative of mature CBF protein, but did not react with a lysate of a ?nmb0345 mutant (CBF-), demonstrating specificity of the murine immune responses. CBF protein was produced by all strains of meningococci studied thus far and the protein was present on the surface of MC58 (CBF+) bacteria, but absent on ?nmb0345 mutant (CBF-) bacteria, as judged by FACS reactivity of anti-rCBF sera. Analysis of the NEIS1825 amino acid sequences from 6644 N. meningitidis isolates with defined Alleles in the pubmlst.org/Neisseria database showed that there were 141 ST types represented and there were 136 different allelic loci encoding 49 non-redundant protein sequences. Only 6/6644 (&lt;0.1%) of N. meningitidis isolates lacked the nmb0345 gene. Amongst serogroup B isolates worldwide, ~68% and ~20% expressed CBF encoded by Allele 1 and 18 respectively, with the proteins sharing &gt;99% amino acid identity. Murine antisera to rCBF in Zw 3–14 micelles + MPLA induced significant serum bactericidal activity (SBA) against homologous Allele 1 and heterologous Allele 18 strains, using both baby rabbit serum complement and human serum complement (h)SBA assays, but did not kill strains expressing heterologous protein encoded by Alelle 2 or 3. Furthermore, variable bactericidal activity was induced by murine antisera against different meningococcal strains in the hSBA assay, which may correlate with variable surface exposure of CBF. Regardless, the attributes of amino acid sequence conservation and protein expression amongst different strains and the ability to induce cross-strain bactericidal antibodies indicates that rCBF could be a potential meningococcal vaccine antigen and merits further testin

Neisseria gonorrhoeae Pilus Attenuates Cytokine Response of Human Fallopian Tube Explants

Background. A role for pilus during attachment of Neisseria gonorrhoeae to epithelia of the female reproductive tract is currently assumed. However, Pil− gonococci have been observed during infection of the reproductive tract, which prompted us to examine the effect of pili on the dynamics of infection and the inflammatory responses of mucosal explants of the human Fallopian tube. Methods. Mucosal explants were infected in vitro with Opa negative Pil− and Pil+N. gonorrhoeae strains. Results. Piliation enhanced gonococcal adherence to the epithelium within 3 h of infection (P < 0.05) but thereafter did not offer advantage to gonococci to colonize the epithelial cell surface (P > 0.05). No differences were found between the strains in numbers of gonococci inside epithelial cells. Pil− bacteria induced higher levels (P < 0.05) of IL-1β, TNF-α, GM-CSF, MCP-1, and MIP-1β than Pil+ bacteria. There were no differences between both strains in LOS pattern, and Pil expression did not change after coincubation with mucosal strips. Conclusions. Results show that gonococcal invasion of the human Fallopian tube can occur independently of pilus or Opa expression, and suggest that pilus, by inhibition of several key elements of the initial inflammatory response, facilitates sustained infection of this organ

Viral inhibition of bacterial phagocytosis by human macrophages: redundant role of CD36

Macrophages are essential to maintaining lung homoeostasis and recent work has demonstrated that influenza-infected lung macrophages downregulate their expression of the scavenger receptor CD36. This receptor has also been shown to be involved in phagocytosis of Streptococcus pneumoniae, a primary agent associated with pneumonia secondary to viral infection. The aim of this study was to investigate the role of CD36 in the effects of viral infection on macrophage phagocytic function. Human monocyte-derived macrophages (MDM) were exposed to H3N2 X31 influenza virus, M37 respiratory syncytial virus (RSV) or UV-irradiated virus. No infection of MDM was seen upon exposure to UV-irradiated virus but incubation with live X31 or M37 resulted in significant levels of viral detection by flow cytometry or RT-PCR respectively. Infection resulted in significantly diminished uptake of S. pneumoniae by MDM and significantly decreased expression of CD36 at both the cell surface and mRNA level. Concurrently, there was a significant increase in IFN? gene expression in response to infection and we observed a significant decrease in bacterial phagocytosis (p = 0.031) and CD36 gene expression (p = 0.031) by MDM cultured for 24 h in 50IU/ml IFN?. Knockdown of CD36 by siRNA resulted in decreased phagocytosis, but this was mimicked by transfection reagent alone. When MDM were incubated with CD36 blocking antibodies no effect on phagocytic ability was observed. These data indicate that autologous IFN? production by virally-infected cells can inhibit bacterial phagocytosis, but that decreased CD36 expression by these cells does not play a major role in this functional deficiency

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Preparation of Lipooligosaccharide (LOS) from Neisseria gonorrhoeae

Neisseria gonorrhoeae is a gram-negative obligate human pathogen that contains lipooligosaccharide (LOS) as a major constituent within the outer membrane. LOS plays a major role in pathogenesis by inducing host inflammatory responses and also enabling evasion of host innate immunity through sialylation. Epitopes within LOS are also potential vaccine candidates. In this chapter, we describe a general method based on the Westphal hot phenol extraction process to purify whole LOS from N. gonorrhoeae for structural analyses and for use in in vivo and in vitro biological assays.</p

Neisseria proteomics for antigen discovery and vaccine development

Neisseria meningitidis (meningococcus) is a major causative organism of meningitis and sepsis and Neisseria gonorrhoeae (gonococcus) is the causative organism of the sexually transmitted disease gonorrhea. Infections caused by meningococci are vaccine-preventable, whereas gonococcal vaccine research and development has languished for decades and the correlates of protection are still largely unknown. In the past two decades, complementary ‘omic’ platforms have been developed to interrogate Neisseria genomes and gene products. Proteomic techniques applied to whole Neisseria bacteria, outer membranes and outer membrane vesicle vaccines have generated protein maps and also allowed the examination of environmental stresses on protein expression. In particular, immuno-proteomics has identified proteins whose expression is correlated with the development of human natural immunity to meningococcal infection and colonization and following vaccination. Neisseria proteomic techniques have produced a catalog of potential vaccine antigens and investigating the functional and biological properties of these proteins could finally provide ‘universal’ Neisseria vaccines.<br/

A DNA vaccine strategy for effective antibody induction to pathogen-derived antigens

DNA-based vaccines are currently being developed for treating a diversity of human diseases including cancers, autoimmune conditions, allergies, and microbial infections. In this chapter, we present a general protocol that can be used as a starting point for developing DNA vaccines to pathogen-derived antigens, using Neisseria meningitidis as an example. In addition, we describe a fusion gene-based vaccine protocol for increasing the potency of DNA vaccines that are based on poorly immunogenic antigens such as short pathogen-derived polypeptides. Finally, we provide a safe and effective protocol for delivery of DNA vaccines, based on intramuscular injection followed by electroporation