Multiple antigen peptide vaccines against Plasmodium falciparum malaria.

The multiple antigen peptide (MAP) approach is an effective method to chemically synthesize and deliver multiple T-cell and B-cell epitopes as the constituents of a single immunogen. Here we report on the design, chemical synthesis, and immunogenicity of three Plasmodium falciparum MAP vaccines that incorporated antigenic epitopes from the sporozoite, liver, and blood stages of the life cycle. Antibody and cellular responses were determined in three inbred (C57BL/6, BALB/c, and A/J) strains, one congenic (HLA-A2 on the C57BL/6 background) strain, and one outbred strain (CD1) of mice. All three MAPs were immunogenic and induced both antibody and cellular responses, albeit in a somewhat genetically restricted manner. Antibodies against MAP-1, MAP-2, and MAP-3 had an antiparasite effect that was also dependent on the mouse major histocompatibility complex background. Anti-MAP-1 (CSP-based) antibodies blocked the invasion of HepG2 liver cells by P. falciparum sporozoites (highest, 95.16% in HLA-A2 C57BL/6; lowest, 11.21% in BALB/c). Furthermore, antibodies generated following immunizations with the MAP-2 (PfCSP, PfLSA-1, PfMSP-1(42), and PfMSP-3b) and MAP-3 (PfRAP-1, PfRAP-2, PfSERA, and PfMSP-1(42)) vaccines were able to reduce the growth of blood stage parasites in erythrocyte cultures to various degrees. Thus, MAP-based vaccines remain a viable option to induce effective antibody and cellular responses. These results warrant further development and preclinical and clinical testing of the next generation of candidate MAP vaccines that are based on the conserved protective epitopes from Plasmodium antigens that are widely recognized by populations of divergent HLA types from around the world.

Antibodies against a multiple-peptide conjugate comprising chemically modified human immunodeficiency virus type-1 functional Tat peptides inhibit infection.

We demonstrated recently that selective side-chain modification of functional cysteine-rich (Tat(21-40)) and arginine-rich (Tat(53-68)) domains of the HIV-1 Tat protein blocks pathogenic activities of these peptides while retaining their immunological characteristics. In the present study, we have synthesized a multiple-peptide conjugate system comprising modified Tat(21-40) and Tat(53-68) peptides (HIV-1-Tat-MPC). Immunization of mice with this highly homogeneous 10.7 kDa HIV-1-Tat-MPC synthetic construct induced an effective immune response in mice. The antibodies generated against HIV-1-Tat-MPC efficiently suppressed Tat-induced viral replication and significantly reduced HIV-associated cytopathic effects in human monocytes. These results indicate that epitope-specific antibodies directed against functional sites of Tat protein using non-pathogenic peptides inhibit HIV pathogenesis. The HIV-1-Tat-MPC, therefore, has potential for the development of a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV infection.

Structural basis of peroxide-mediated changes in human hemoglobin: a novel oxidative pathway.

Hydrogen peroxide (H(2)O(2)) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H(2)O(2) to highly purified human hemoglobin (HbA(0)) induced structural changes that primarily resided within beta subunits followed by the internalization of the heme moiety within alpha subunits. These modifications were observed when an equal molar concentration of H(2)O(2) was added to HbA(0) yet became more abundant with greater concentrations of H(2)O(2). Mass spectrometric and amino acid analysis revealed for the first time that betaCys-93 and betaCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA(0) was treated with H(2)O(2). Oxidation of further amino acids in HbA(0) exclusive to the beta-globin chain included modification of betaTrp-15 to oxyindolyl and kynureninyl products as well as betaMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the beta subunits as a result of the H(2)O(2) attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two alpha-peptide fragments (alpha128-alpha139) and a heme moiety with the loss of iron, cross-linked between alphaSer-138 and the porphyrin ring. The novel oxidative pathway of HbA(0) modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.

Chemical characterization of diaspirin cross-linked hemoglobin polymerized with poly(ethylene glycol).

A lack of specificity associated with chemical modification methods used in the preparation of certain hemoglobin (Hb)-based oxygen carriers (HBOCs) may alter Hb structure and function, as amino acids located in critical regions (e.g., alpha-beta interfaces and the 2,3-DPG binding pocket) may unintentionally be targeted. Hb protein surface modifications with various poly(ethylene glycol) (PEG) derivatives have been used as conjugating and polymerizing agents with the intent of improving reaction site specificity/reproducibility and ultimately reducing the untoward hypertensive response due to nitric oxide scavenging by smaller molecular size tetrameric species (i.e., 64 kDa) in HBOC solutions. Previous experiments performed in our laboratory have evaluated the influence of polymerization of diaspirin alpha-alpha cross-linked Hb (alphaalpha-DBBF-Hb) with a bifunctional modified PEG, bis(maleoylglycylamide) PEG (BMAA-PEG), in terms of oxygen carrying capacity, redox properties, hypertensive response, and renal clearance in rats. The data presented in this paper specifically evaluate the influence of BMAA-PEG on alphaalpha-DBBF-Hb (Poly-alphaalpha-DBBF-Hb) to identify molecular weight distribution, protein conformation, and site-specific modification, as well as to provide insight into the previously determined in vitro and in vivo functional and vasoactive characteristics of this HBOC. Chemical analysis performed herein reveals nonspecific modifications induced by BMAA-PEG that result in the full modification of alphaalpha-DBBF-Hb leaving no tetrameric cross-linked starting material in solution. These data are inconsistent with the continuing assumption that molecular size (i.e., 64 kDa) has a direct influence on HBOC-mediated vasoactivity and that other protective strategies should be considered to control blood pressure imbalances.

CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin.

CD163 mediates the internalization of hemoglobin-haptoglobin (Hb-Hp) complexes by macrophages. Because Hp binding capacity is exhausted during severe hemolysis, an Hp-independent Hb-clearance pathway is presumed to exist. We demonstrate that Hb interacts efficiently with CD163 in the absence of Hp. Not only is Hb internalized into an endosomal compartment by CD163 as a result of active receptor-dependent endocytosis; it also inhibits the uptake of Hb-Hp complexes, suggesting a common receptor-binding site. Free Hb further induces heme oxygenase mRNA expression in CD163+ HEK293 cells, but not in CD163- cells. Additional evidence for Hp-independent Hb-CD163 interaction is provided by the demonstration that CD163 mediates the uptake of alpha alpha-DBBF crosslinked Hb, a chemically modified Hb that forms minimal Hp complexes. Moreover, certain modifications to Hb, such as polymerization or the attachment of specific functional groups (3 lysyl residues) to the beta-Cys93 can reduce or enhance this pathway of uptake. In human macrophages, Hp-complex formation critically enhances Hb uptake at low (1 microg/mL), but not at high (greater than 100 microg/mL), ligand concentrations, lending support for a concentration-dependent biphasic model of macrophage Hb-clearance. These results identify CD163 as a scavenger receptor for native Hb and small-molecular-weight Hb-based blood substitutes after Hp depletion.

Genetic and functional analyses of the lgtH gene, a member of the beta-1,4-galactosyltransferase gene family in the genus Neisseria.

Lipooligosaccharide (LOS) is a major virulence factor of the pathogenic Neisseria. Three galactosyltransferase genes, lgtB, lgtE and lgtH, responsible for the biosynthesis of LOS oligosaccharide chains, were analysed in five Neisseria species. The function of lgtH in Neisseria meningitidis 6,275 was determined by mutagenesis and chemical characterization of the parent and mutant LOS chains. The chemical characterization included SDS-PAGE, immunoblot, hexose and mass spectrometry analyses. Compared with the parent LOS, the mutant LOS lacked galactose, and its oligosaccharide decreased by three or four sugar units in matrix-assisted laser desorption ionization (MALDI)-MS analysis. The results show that lgtH encodes a beta-1,4-galactosyltransferase, and that the glucose moiety linked to heptose (Hep) in the alpha chain is the acceptor site in the biosynthesis of Neisseria LOS. To understand the sequence diversity and relationships of lgtB, lgtE and lgtH, the entire lgt-1 locus was further sequenced in three N. meningitidis strains and three commensal Neisseria strains, and compared with the previously reported lgt genes from Neisseria species. Comparison of the protein sequences of the three enzymes LgtB, LgtE and LgtH showed a conserved N-terminal region, and a highly variable C-terminal region, suggesting functional constraint for substrate and acceptor specificity, respectively. The analyses of allelic variation and evolution of 23 lgtB, 12 lgtE and 14 lgtH sequences revealed a distinct evolutionary history of these genes in Neisseria. For example, the splits graph of lgtE displayed a network evolution, indicating frequent DNA recombination, whereas splits graphs of lgtB and lgtH displayed star-tree-like evolution, indicating the accumulation of point mutations. The data presented here represent examples of the evolution and variation of prokaryotic glycosyltransferase gene families. These imply the existence of multiple enzyme isoforms for biosynthesis of a great diversity of oligosaccharides in nature.

Selective side-chain modification of cysteine and arginine residues blocks pathogenic activity of HIV-1-Tat functional peptides.

Extracellular Tat protein of HIV-1 activates virus replication in HIV-infected cells and induces a variety of host factors in the uninfected cells, some of which play a critical role in the progression of HIV infection. The cysteine-rich and arginine-rich basic domains represent key components of the HIV-Tat protein for pathogenic effects of the full-length Tat protein and, therefore, could be ideal candidates for the development of a therapeutic AIDS vaccine. The present study describes selective modifications of the side-chain functional groups of cysteine and arginine amino acids of these HIV-Tat peptides to minimize the pathogenic effects of these peptides while maintaining natural peptide linkages. Modification of cysteine by introducing either a methyl or t-butyl group in the free sulfhydryl group and replacing the guanidine group with a urea linkage in the side chain of arginine in the cysteine-rich and arginine-rich Tat peptide sequences completely blocked the ability of these peptides to induce HIV replication, chemokine receptor CCR-5 expression, and NF-kappaB activity in monocytes. Such modifications also inhibited angiogenesis and migration of Kaposi's sarcoma cells normally induced by Tat peptides. Such chemical modifications of the cysteine-rich and arginine-rich peptides did not affect their reactivity with antibodies against the full-length Tat protein. With an estimated 40 million HIV-positive individuals worldwide and approximately 4 million new infections emerging every year, a synthetic subunit HIV-Tat vaccine comprised of functionally inactive Tat domains could provide a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV disease.

Structural and functional characterization of glutaraldehyde-polymerized bovine hemoglobin and its isolated fractions.

Glutaraldehyde-polymerized bovine hemoglobin (PolyHbBv, trade name Oxyglobin), is a non-site-specific modified hemoglobin-based oxygen-carrying solution, developed for use in veterinary medicine. PolyHbBv was fractionated into four distinct tetrameric and multiple polytetrameric forms ranging in molecular mass (87.2-502.3 kDa) using size exclusion chromatography (SEC) and verified by laser light scattering. We evaluated the structural modification occurring in the fractionated mixture of PolyHbBv and assessed the functionality and redox stability of each fraction in relation to the mixture as a whole. Intramolecular cross-linking evaluation as performed by MALDI-MS and SEC under dissociating conditions revealed no-site-specific tetramer stabilization within the fractions; Intermolecular cross-linking was highly correlated with lysine and histidine modification as determined by amino acid composition analysis. While native unmodified hemoglobin, HbBv, PolyHbBv, and PolyHbBv fractions (F1-F4) revealed significant methionine oxidation, modification, or both, the critical betaMet55 located in the functionally plastic domains (alpha1-beta1 interface) of HbBv was unaltered. Moreover, neither of the two betaCys93 located in the highly plastic alpha1-beta2 interface were modified in PolyHbBv or in F1-F4. Our structural analysis also revealed that the reported loss in sensitivity to chloride in PolyHbBv could not be attributed to direct alteration of chloride ion binding amino acids. Structural modification imparted by glutaraldehyde resulted in nearly identical functional characteristics of PolyHbBv and its fractions with regard to oxygen equilibrium, ligand binding, and autoxidative kinetics.

O-raffinose crosslinked hemoglobin lacks site-specific chemistry in the central cavity: structural and functional consequences of beta93Cys modification.

Reacting human deoxyHbA0 with oxidized raffinose (O-raffinose), a trisaccharide, results in a low oxygen affinity "blood substitute," stabilized in a noncooperative T-conformation and possesses readily oxidizable rhombic heme. In this study, we fractionated the O-raffinose-modified HbA0 heterogeneous polymer (O-R-PolyHbA0) into six distinct fractions with a molecular weight distribution ranging from 64 to approximately 600 kDa using size-exclusion chromatography (SEC). Oxygen equilibrium and kinetics binding parameters of all fractions were nearly identical, reflecting a lack of heterogeneity in ligand binding properties among O-R-PolyHbA0 species (Hill coefficient n equal to 1.0). Several mass spectrometry techniques were used to evaluate undigested and digested HbA0, O-R-PolyHbA0, and O-R-PolyHbA0 fractions. Proposed sites of intramolecular crosslinking (i.e., beta1Lys82, beta2Lys82, and beta1Val1) were not found to be the predominant site of crosslinking within the central cavity. Intermolecular crosslinking with O-raffinose results in no discernible site of amino acids modifications with the exception of beta93Cys and alpha104Cys. Based on accessible surface area (ASA) calculations in intact deoxyHbA0, slight conformational changes are required to allow for the S on alpha104Cys to be modified during the reaction with O-raffinose or its partially oxidized product(s). The stabilization of HbA0 in the T-conformation may not be a direct correlate of O-raffinose induced changes, but an indirect consequence of changing hydration in the water-filled central cavity and/or the distal heme pocket leading in the latter case to accelerated iron oxidation. Structural data presented here when taken together with the oxidative instability of O-R-PolyHbA0 may provide some basis for the reported toxicity of this oxygen carrier.

Susceptibility of the hydroxyl groups in serine and threonine to beta-elimination/Michael addition under commonly used moderately high-temperature conditions.

The beta-elimination/Michael addition reaction has been employed for the modification of O-acylated and phosphorylated Ser and Thr residues in a variety of derivatives. The modified Ser and Thr can be analyzed by amino acid composition analysis, N-terminal Edman degradation sequence analysis, and tandem mass spectrometric sequencing which generally allows the identification and localization of the phosphorylation or glycosylation sites. However, the reactivity of the free hydroxyl group on serine and threonine by sodium hydroxide-induced beta-elimination has not been critically examined. In this study, two analogous phosphopeptides, KMpSTLSYR and KMSpTLSYR, were subjected to beta-elimination under the widely used conditions previously reported, followed by sulfite or ethanethiol addition. After treatment of the phosphopeptides in 0.1 N NaOH/0.6 M Na(2)SO(3) at 37 degrees C for 24 h, matrix-assisted laser desorption ionization-time of flight mass spectrometric analyses of the products revealed an appreciable mass peak with an additional observed mass of 64 compared to the expected mass from the conversion of phosphate to sulfite. Similarly, treatment of the phosphopeptides in 0.52 N NaOH/1.36 M ethanethiol at 50 degrees C for 18 h or for even as short as 1h also yielded additional 44 mass of ethylthiogroup in excess of the expected mass for the modified phosphopeptide. Electrospray ionization tandem mass spectrometric analysis confirms that the modification occurred on the hydroxyl group of Ser and Thr in addition to P-Ser and P-Thr. On the other hand, modification on the free hydroxyl group of Ser or Thr was not detected under the mild condition of 0.1 N NaOH/0.6 M Na(2)SO(3) at 25 degrees C for 24 h as previously reported. This finding suggests that temperatures above 25 degrees C and excessive alkalinity should be avoided to prevent the beta-elimination of the hydroxyl group of Ser and Thr in peptides. This is of particular concern when employing highly sensitive tandem mass spectrometric methods for the identification and localization of Ser and Thr as modification sites by the beta-elimination/Michael addition reaction. The additional modification site(s) may complicate the interpretation of data and lead to an erroneous conclusion.

Identification of phosphoserine and phosphothreonine as cysteic acid and beta-methylcysteic acid residues in peptides by tandem mass spectrometric sequencing.

Tandem mass spectrometry has long been an intrinsic tool to determine phosphorylation sites in proteins. However, loss of the phosphate moiety from both phosphoserine and phosphothreonine residues in low-energy collision-induced dissociation is a common phenomenon, which makes identification of P-Ser and P-Thr residues complicated. A method for direct sequencing of the Ser and Thr phosphorylation sites by ESI tandem mass spectrometry following beta-elimination/sulfite addition to convert -HPO4 to -SO3 has been studied. Five model phosphopeptides, including three synthetic P-Ser-, P-Thr-, or P-Ser- and P-Thr-containing peptides; a protein kinases C-phosphorylated peptide; and a phosphopeptide derived from beta-casein trypsin digests were modified and then sequenced using an ESI-quadrupole ion trap mass spectrometer. Following incubation of P-Ser- or P-Thr-containing peptides with Na2SO3/NaOH, 90% P-Ser and 80% P-Thr was converted to cysteic acid and beta-methylcysteic acid, respectively, as revealed by amino acid analysis. The conversion can be carried out at 1 microM concentration of the peptide. Both cysteic acid and beta-methylcysteic acid residues in the sequence were shown to be stable and easily identifiable under general conditions for tandem mass spectrometric sequencing applicable to common peptides.