Immunogenicity in rhesus of the Plasmodium vivax mosquito stage antigen Pvs25H with Alhydrogel and Montanide ISA 720.

Pvs25 is an ookinete surface protein from Plasmodium vivax that is the target of transmission-blocking antibodies. Two immunogenicity trials in rhesus monkeys with a recombinant form of the protein, Pvs25H, were undertaken. Monkeys were vaccinated with Pvs25H adsorbed to Alhydrogel or emulsified in Montanide ISA 720 at 0, 4 and 27 weeks (study 1) or in Montanide ISA 720 at 0 and 18 weeks (study 2) with 1.5 or 15 microg Pvs25H in 0.1 or 0.5 mL of emulsion (four combinations). Immunogenicity was assessed by ELISA and by membrane-feeding experiments using P. vivax-infected blood from human volunteers (studies 1 and 2) or from chimpanzees (study 1). Both vaccine trials generated antibodies that blocked transmission of P. vivax to mosquitoes. Antibody titres and transmission blocking were higher with Montanide ISA 720 than with Alhydrogel in the first trial and with the 15 microg Pvs25H/0.5 mL ISA 720 combination in the second trial.

Disulfide bonds in merozoite surface protein 1 of the malaria parasite impede efficient antigen processing and affect the in vivo antibody response.

Vol. 34(3) 2004, DOI 10.1002/eji.200324514 Due to a technical error, the wrong affiliations were given for C. Moss and V. Lindo. These are correct as given above. See original article http://dx.doi.org/10.1002/eji.200324514.

Cellular basis of early cytokine response to Plasmodium falciparum.

Uncertainty remains about the cellular origins of the earliest phase of the proinflammatory cytokine response to malaria. Here we show by fluorescence-activated cell sorter analysis that gammadelta T cells and CD14+ cells from nonimmune donors produce tumor necrosis factor and that gammadelta T cells also produce gamma interferon within 18 h of contact with mycoplasma-free Plasmodium falciparum-infected erythrocytes in vitro. This early cytokine response is more effectively induced by intact than by lysed parasitized erythrocytes. However, the IFN-gamma response to lysed parasites is considerably enhanced several days after peripheral blood mononuclear cells are primed with low numbers of intact parasitized erythrocytes, and in this case it derives from both alphabeta and gammadelta T cells. These data show that naïve gammadelta T cells can respond very rapidly to malaria infection but that malaria fever may involve a multistage process in which the priming of both gammadelta and alphabeta T-cell populations boosts the cytokine response to lysed parasite products released at schizont rupture.

Early cytokine induction by Plasmodium falciparum is not a classical endotoxin-like process.

We have investigated the widely held view that malaria parasites induce pro-inflammatory cytokines primarily through an endotoxin-like stimulatory effect on macrophages. We report that the pattern of cytokine production by non-immune human peripheral blood mononuclear cells following stimulation by Plasmodium falciparum-infected erythrocytes (Pfe) in vitro differs considerably from that induced by bacterial endotoxin. The Pfe-induced TNF response at day 1 is associated with a much higher level of IFN-gamma production and a much lower level of IL-12 p40 and IL-10 expression than a comparable endotoxin-induced TNF response. Both CD3(+) and CD14(+) populations are required for this early TNF response to Pfe, whereas the endotoxin-induced response is unaffected by depletion of the CD3(+) population. Pfe fails to stimulate the monocyte-like cell line MonoMac6 to express pro-inflammatory cytokines. These findings suggest that the early inflammatory response to malaria is critically dependent on lymphocyte subpopulations that play a lesser role in the response to bacterial endotoxin.

Alternative modes of tyrosyl phosphopeptide binding to a Src family SH2 domain: implications for regulation of tyrosine kinase activity.

Src homology 2 (SH2) domains interact with proteins containing phosphorylated tyrosine residues and as such play a key role in mediating tyrosine kinase signal transduction. Determination of how these interactions maintain specificity is central to understanding the mechanism of this intracellular signal processing. In the Src family tyrosine kinases specificity is enhanced by a form of regulation based on binding of a phosphotyrosine, pY, and its proximal amino acid sequence from the C-terminus to the SH2 domain of the same protein (autoregulation) or to a similar protein (homodimeric regulation). Activation of the protein is accomplished by removal of this regulatory interaction by competition from a "specific" interacting ligand. We adopt the SH2 domain from a member of the Src family, Fyn (whose predominant physiological role is in initiation of signals from the T-cell receptor complex), to explore the differences in structural, thermodynamic, and kinetic determinants of regulatory and specific interactions using tyrosyl phosphopeptides based on the C-terminus and on a putative physiological interacting species from the hamster middle-sized tumor antigen. The specific peptide interacts with micromolar affinity via embedding the pY and an isoleucine residue (in the pY + 3 position) in two deep pockets. This leads to a large favorable enthalpic contribution to free energy. The regulatory peptide interacts in the pY pocket which forms a pivot for the rest of the molecule which is dynamic. These structural data for the regulatory peptide are supported by the observation of a more favorable entropic term and a complex mode of binding revealed by kinetic analysis.

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure, backbone dynamics and protein association.

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155-270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of 1H-15N-correlated 2D HSQC, 15N-edited 3D TOCSY-HMQC, and 15N-edited 3D NOESY-HMQC spectra. By analysis of the alpha-proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain. To investigate the internal dynamics of the protein backbone, T1 and T2 relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.

Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.

The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study.