Using the PfEMP1 Head Structure Binding Motif to Deal a Blow at Severe Malaria

Plasmodium falciparum (Pf) malaria causes 200 million cases worldwide, 8 million being severe and complicated leading to similar to 1 million deaths and similar to 100,000 abortions annually. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) has been implicated in cytoadherence and infected erythrocyte rosette formation, associated with cerebral malaria; chondroitin sulphate-A attachment and infected erythrocyte sequestration related to pregnancy-associated malaria and other severe forms of disease. An endothelial cell high activity binding peptide is described in several of this similar to 300 kDa hypervariable protein's domains displaying a conserved motif (GACxPxRRxxLC); it established H-bonds with other binding peptides to mediate red blood cell group A and chondroitin sulphate attachment. This motif (when properly modified) induced PfEMP1-specific strain-transcending, fully-protective immunity for the first time in experimental challenge in Aotus monkeys, opening the way forward for a long sought-after vaccine against severe malaria

Identification and characterization of the Plasmodium vivax thrombospondin-related apical merozoite protein

<p>Abstract</p> <p>Background</p> <p>Malaria caused by <it>Plasmodium vivax </it>is a major public health problem worldwide that affects 70-80 million people in the Middle East, Asia, Western Pacific, South America and the Caribbean. Despite its epidemiological importance, few antigens from this parasite species have been characterized to date compared to <it>Plasmodium falciparum</it>, due in part to the difficulties of maintaining an <it>in vitro </it>culture of <it>P. vivax</it>. This study describes the identification of the <it>P. falciparum </it>thrombospondin-related apical merozoite protein homologue in <it>P. vivax </it>(PvTRAMP) and examines its potential to be further evaluated as vaccine candidate.</p> <p>Methods</p> <p>The gene encoding PvTRAMP was identified through an extensive search of the databases hosting the genome sequence of <it>P. vivax</it>. Genes adjacent to <it>pvtramp </it>were identified <it>in silico </it>to determine the degree of similarity between the protein sequences encoded by equivalent chromosomic fragments in <it>P. falciparum </it>and <it>Plasmodium knowlesi</it>. The <it>pvtramp </it>gene was amplified from cDNA of <it>P. vivax </it>schizont stages, cloned and expressed in <it>Escherichia coli</it>. Anti-PvTRAMP antisera was obtained by inoculating rabbits with PvTRAMP B cell epitopes produced as synthetic peptides in order to assess its recognition in parasite lysates by Western blot and in intact parasites by indirect immunofluorescence. The recognition of recombinant PvTRAMP by sera from <it>P. vivax-</it>infected individuals living in endemic areas was also assessed by ELISA.</p> <p>Results</p> <p>The PfTRAMP homologue in <it>P. vivax</it>, here denoted as PvTRAMP, is a 340-amino-acid long antigen encoded by a single exon that could have a potential role in cytoadherence, as indicated by the presence of a thrombospondin structural homology repeat (TSR) domain. According to its transcription and expression profile, PvTRAMP is initially located at the parasite's apical end and later on the parasite surface. Recombinant PvTRAMP is recognized by sera from infected patients, therefore, indicating that it is targeted by the immune system during a natural infection with <it>P. vivax.</it></p> <p>Conclusions</p> <p>The results of this work support conducting further studies with PvTRAMP to evaluate its immunogenicity and protection-inducing ability in the <it>Aotus </it>animal model.</p

Vaccine potential of Adhesin Complex Protein (ACP) from <i>Neisseria gonorrhoeae</i>

Sexual transmitted disease Gonorrhoea is caused by the organism Neisseria gonorrhoeae, infecting ~106 million cases annually. Currently, a lack of an effective vaccine against this pathogen and treatments using last generation of antibiotics are misleading due to emerging antibiotic-resistant superbugs. Recently has been described a small protein in a closed related bacteria, N. meningitidis (Nm), termed as an Adhesin Complex Protein (ACP). Nm-ACP is an outer-membrane protein which is a conserved protein in commensal and pathogenic bacteria. Besides Nm-ACP is capable of induce cross-protective bactericidal antibodies. A homologue gene, ng-acp (NGO1981) from Neisseria gonorrhoeae strain P9-17 is highly conserved among gonococcal isolates reported until the date. The ng-acp gene product was cloned into pRSET-A and pET-22b cloning vector systems and expressed as a recombinant protein in E. coli BL21pLysS to be used in immunization trials in murine model using a range of adjuvants and delivery formulations. Raised mice serum demonstrated a great reactivity against recombinant rNg-ACP by ELISA and displayed cross-strain reactivity in gonococcal outer-membrane (OMV) and lysate preparations from N. gonorrhoeae strains P9-17 and FA1090 by western-blot. Antisera r-Ng-ACP showed high bactericidal properties against homologous and heterologous wild type strains compared to the knockout strains. Furthermore, Ng-ACP plays a role of association on different epithelial cells showing a reduction ̴75-50% by comparison the wild-type and knockout. Three–dimensional structure of rNgACP the overall fold resemble of the lysozyme inhibitors from Salmonella typhimurium (PliC family) and recently described Neisseria meningitidis.Taken all together, suggest that Ng-ACP from N. gonorrhoeae is a potential candidate to develop an anti-gonococcal vaccine

Basic methods for examining <i>Neisseria gonorrhoeae</i> interactions with host cells in vitro

The obligate human pathogen Neisseria gonorrhoeae colonizes primarily the mucosal columnar epithelium of the male urethra and the female endocervix. In addition, gonococci can infect the anorectal, pharyngeal, and gingival mucosae and epithelial cells of the conjunctiva. More rarely, the organism can disseminate through the bloodstream, which can involve interactions with other host cell types, including blood vessel endothelial cells and innate immune cells such as dendritic cells, macrophages, and neutrophils. “Disseminated gonococcal infection” is a serious condition with various manifestations resulting from the seeding of organs and tissues with the pathogen. The host response to gonococcal infection is inflammatory. Knowledge of the biology of gonococcal interactions has been served well through the use of a wide variety of ex vivo models using host tissues and eukaryotic cell monocultures. These models have helped identify bacterial surface adhesins and invasins and the corresponding cell surface receptors that play roles in gonococcal pathogenesis. Furthermore, they have been useful for understanding virulence mechanisms as well as innate and adaptive immune responses. In this chapter, readers are provided with protocols for examining the basic interactions between gonococci and a representative human cell line.</p

Gauche+ side-chain orientation as a key factor in the search for an immunogenic peptide mixture leading to a complete fully protective vaccine

Topological and stereo-electron characteristics are essential in major histocompability class II-peptide-T-cell receptor (MHC-p-TCR) complex formation for inducing an appropriate immune response. Modified high activity binding peptides (mHABPs) were synthesised for complete full protection antimalarial vaccine development producing a large panel of individually fully protection-inducing protein structures (FPIPS) and very high long-lasting antibody-inducing (VHLLAI) mHABPs. Most of those which did not interfere, compete, inhibit or suppress their individual VHLLAI or FPIPS activity contained or displayed a polyproline II-like (PPIIL) structure when mixed. Here we show that amino acid side-chains located in peptide binding region (PBR) positions p3 and p7 displayed specific electron charges and side-chain gauche+ orientation for interacting with the TCR. Based on the above, and previously described physicochemical principles, non-interfering, long-lasting, full protection-inducing, multi-epitope, multistage, minimal subunit-based chemically synthesised mHABP mixtures can be designed for developing vaccines against diseases scourging humankind, malaria being one of them. © 2014 Elsevier Ltd

Vaccine potential of bacterial macrophage infectivity potentiator (MIP)-like peptidyl prolyl cis/trans isomerase (PPIase) proteins

Peptidyl prolyl cis/trans isomerases (PPIases) are a superfamily of proteins ubiquitously distributed among living organisms, which function primarily to assist the folding and structuring of unfolded and partially folded polypeptide chains and proteins. In this review, we focus specifically on the Macrophage Infectivity Potentiator (MIP)-like PPIases, which are members of the immunophilin family of FK506-binding proteins (FKBP). MIP-like PPIases have accessory roles in virulence and are candidates for inclusion in vaccines protective against both animal and human bacterial pathogens. A structural vaccinology approach obviates any issues over molecular mimicry and potential cross-reactivity with human FKBP proteins and studies with a representative antigen, the Neisseria meningitidis-MIP, support this strategy. Moreover, a dual approach of vaccination and drug targeting could be considered for controlling bacterial infectious diseases of humans and animals.</p

Structural characterization of HABPs present in crystallized Duffy binding like domains (DBL).

<p>DBL domain 3D structure determined by X-ray crystallography A) Head structure: DBL1α (PDB 2XU0) (pink), C) DBL3X (PDB 3CML) (yellow), F) DBL6ε (PDB 2WAU) (pale blue). ¹H-NMR-determined structure localisation, displaying the perfect fit of HABP 6505 (yellow) superimposed onto DBL1α, 6583 (dark blue) and 6584 (purple) onto DBL3X and 6622 (grey) onto DBL6ε. B, D, G). H-bonds between HABP residues and their corresponding sequence on top, displaying relevant residues in binding to A blood group trisaccharides and CSPG (asterisk and black dot, respectively). E) Superimposed conserved binding motif fragments from 6510 and 6573. H) CD spectra for corresponding HABPs.</p

Humoral immune response and protective efficacy induced by <i>Pf</i>EMP1 HABPs derived peptides in <i>Aotus</i> monkeys.

<p><i>Aotus</i> monkeys’ humoral immune responses and protective immunity induced by <i>Pf</i>EMP1-derived peptides, according to our serial numbering system with corresponding amino acid sequence (modifications in bold). Reciprocal IFA antibody titres in bleeding 20 days after second (II₂₀) and third (III₂₀) immunisation and number of protected monkeys in experimental challenge <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088420#pone.0088420-Patarroyo1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088420#pone.0088420-Curtidor2" target="_blank">[14]</a>.</p

Identification of <i>Pf</i>EMP1 HABPs and variability sequence between <i>Plasmodium falciparum</i> strains.

<p>(<b>A</b>) Dd2 <i>Pf</i>EMP1-based amino-acid sequence synthetic peptides’ RBC and C32 cell binding activity (black bars represent specific binding activity slope); above 2% (dotted line) were considered HABPs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088420#pone.0088420-Rodriguez1" target="_blank">[11]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088420#pone.0088420-Curtidor2" target="_blank">[14]</a>. Blue shows HABPs chosen for immunization and red those containing canonical or homologous (GACxPxRRxxLC) binding motif. Left, schematic representation of <i>Pf</i>EMP1 domains showing H-bonds between HABPs (arrows); head structure recombinant fragments containing NTS and DBL1α (fuchsia), CDR1α (green), DBL3X (orange) and DBL6ε (blue), 3D structure determined by X-ray crystallography. (<b>B</b>) Sequence logos for amino acid conservation in corresponding HABPs according to their frequency in >100 strains; each amino acid height reflects their relative frequency (%) and thus their contribution to conservation.</p