Therapeutic effect of an altered peptide ligand derived from heat-shock protein 60 by suppressing of inflammatory cytokines secretion in two animal models of rheumatoid arthritis.

Rheumatoid arthritis is a systemic autoimmune disease mediated by T cells. Productive engagement of T cell receptors by major histocompatibility complex-peptide leads to proliferation, differentiation and the definition of effector functions. Altered peptide ligands (APL) generated by amino acid substitutions in the antigenic peptide have diverse effects on T cell response. We predicted a novel T cell epitope from human heat-shock protein 60, an autoantigen involved in the pathogenesis of rheumatoid arthritis. Three APLs were designed from this epitope and it was demonstrated that these peptides induce the activation of T cells through their ability to modify cell cycle phase's distribution of CD4+T cells from RA patients. Also, IL-17, TNF-α and IL-10 levels were determined in PBMC from these patients. Unlike the wild-type peptide and the other two APLs, APL2 increased the IL-10 level and suppressed IL-17 secretion in these assays. Therapeutic effect of this APL in adjuvant arthritis (AA) and collagen-induced arthritis (CIA) models was also evaluated. Clinical score, histopathology, inflammatory and regulatory cytokine concentration were monitored in the animals. APL2 efficiently inhibited the progression of AA and CIA with a significant reduction of the clinical and histopathologic score. Therapeutic effect of APL2 on CIA was similar to that obtained with MTX; the standard treatment for RA. This effect was associated with a decrease of TNF-α and IL-17 levels. These results suggest that the therapeutic effect of APL2 is mediated in part by down-regulation of inflammatory cytokines and support the potential use of APL2 as a therapeutic drug in RA patients.

Profiling the immune responses of human patients treated with recombinant streptokinase after myocardial infarct.

The SPOT synthesis of peptide arrays on continuous cellulose membranes should be generally applicable in the analysis of sequential antibody binding sites using the enzyme-substrate or other standard detection protocols. The use of total serum is limited by the occurrence of high background levels. This may be overcome if affinity purified antibodies or sera with high antibody titers are used, which allows work at high dilutions and a consequent reduction of background level. Here we demonstrate the mapping of antigenic regions located on recombinant streptokinase SK-2 (Heberkinase) using cellulose-bound peptide scans and human total sera from patients treated with SK-2 (Heberkinase). Streptokinase (SK) is a 47 kDa protein produced by various strains of hemolytic streptococci and is a potent activator of the fibrinolytic enzyme system in humans. SK is in widespread clinical use to treat acute infarction because of its function as an activator of vascular fibrinolysis. Since streptococcal infections are common, normal individuals are immunized with SK and antibodies (Abs) to SK can be detected in most of them. This therapy generates significant T-cell responses to SK and the neutralizing capacity of the Abs rises significantly. Neutralizing Abs reduces the efficiency of thrombolytic therapy and may cause allergic reactions. The widespread use of SK in humans makes its antigenicity an important clinical problem. In this regard the study of the immunodominant regions of SK becomes an important aspect for the improvement of this thrombolytic agent.

Differential regulation of Th1/Th2 in relevant tissues for sepsis pathogenesis with a Limulus anti-LPS factor-derived peptide increases survival in Gram-positive sepsis.

Severe sepsis and septic shock are important causes of death in intensive care units. Although Gram-negative infections were predominant in the 1960s, Gram-positive infections have increased in the past two decades and now account for about half of the cases of severe sepsis. In this study, we examined the effect of a Limulus anti-LPS factor (LALF)-derived peptide on lung and liver Th1/Th2 cytokine mRNA levels during a Gram-positive sepsis. We also examined the morphopathological changes observed in these organs during the disease. Mice challenged with a high dose of Staphylococcus haemolyticus showed severe damage in lung. In contrast, the liver of challenged mice showed an accumulation of bacterial particles in the sinusoids, associated with a severe inflammatory response due to high levels of tissue mRNA proinflammatory cytokines. Treatment with the peptide LALF(32-51) ameliorated the sepsis-induced effects in the lung and liver and increased the survival of mice in a dose- and time-dependent manner. Pretreatment with the peptide LALF(32-51) differentially regulates TNF-alpha, IFN-gamma, IL-12p40, IL-2 and IL-10 mRNA levels in lung and liver of peptide-treated mice, and limits the systemic inflammatory response. These findings support for the first time the effectiveness of an LALF-derived peptide in the treatment of a Gram-positive sepsis. Modulation of the Th1/Th2 pattern in tissues relevant for sepsis correlates with an improved outcome of the disease as denoted by increased survival.

A Limulus anti-LPS factor-derived peptide modulates cytokine gene expression and promotes resolution of bacterial acute infection in mice.

Sepsis in experimental animals and humans has been associated with perturbed immune response. A major event contributing to the decrease in immune functions in septic disorders seems to be the inadequate balance of cytokines mediating the interactions between the innate and adaptive immune systems. We previously observed that a cyclic peptide derived from the Limulus anti-LPS factor (LALF), which partially protect mice from endotoxic shock lethality, has the ability to modulate cytokine secretion in vitro. We herein examined the effects of the LALF(31-52) peptide in an experimental model of Gram-negative peritoneal sepsis and analyzed the cytokine gene expression in the spleen and liver of peptide-treated mice. The prophylactic administration of LALF(31-52) abrogated the systemic TNF-alpha response, reduced organ damage and increased the survival of infected mice. Histological examination of spleen and liver in peptide-treated mice showed prevention of tissue damage induced by the high dose of Pseudomonas aeruginosa. This treatment modulates the cytokine gene expression in these tissues, stimulating IL-2, IL-12 and IL-13 mRNA synthesis, while IL-4 and IL-10 mRNA expression was not modified. This cytokine profile induced by the LALF-derived peptide seems to be favorable for host resistance against Gram-negative bacteria acute infection. In addition, peptide treatment was effective after the initiation of the systemic inflammatory response, promoting a significant increase in mice survival. These results further demonstrate the immunomodulatory potential of LALF(31-52) and are relevant for the design of prophylactic and therapeutic strategies for acute bacteria infection and sepsis, especially for preventing or ameliorating host immunity defects in these disorders.

Identification of single amino acid residues essential for the binding of lipopolysaccharide (LPS) to LPS binding protein (LBP) residues 86-99 by using an Ala-scanning library.

Lipopolysaccharide binding protein (LBP) is a 60 kDa acute phase glycoprotein capable of binding to LPS of Gram-negative bacteria and facilitating its interaction with cellular receptors. This process is thought to be of great importance in systemic inflammatory reactions such as septic shock. A peptide corresponding to residues 86-99 of human LBP (LBP86-99) has been reported to bind specifically with high affinity the lipid A moiety of LPS and to inhibit the interaction of LPS with LBP. We identified essential amino acids in LBP86-99 for binding to LPS by using a peptide library corresponding to the Ala-scanning of human LBP residues 86-99. Amino acids Trp91 and Lys92 were indispensable for peptide-LPS interaction and inhibition of LBP-LPS binding. In addition, several alanine-substituted synthetic LBP-derived peptides inhibited LPS-LBP interaction. Substitution of amino acids Arg94, Lys95 and Phe98 by Ala increased the inhibitory effect. The mutant Lys95 was the most active in blocking LPS binding to LBP. These findings emphasize the importance of single amino acids in the LPS binding capacity of small peptides and may contribute to the development of new drugs for use in the treatment of Gram-negative bacterial sepsis.

Study of different coupling agents in the conjugation of a V3-based synthetic MAP to carrier proteins.

The conjugation of synthetic peptides to carrier proteins is a widely used method for immunological studies. Different coupling agents have been described to form the conjugate with carrier proteins. In this paper, we demonstrate that the antibody response toward V3-based synthetic MAPs derived from HIV-1, JY1 isolate, conjugated to two different carrier proteins using either m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or beta-maleimidopropionic acid N-hydroxysuccinimide ester (MPS), or succinic anhydride (SA) show different behaviors. An excellent anti-JY1 response without a strong response to the coupling agent is observed in the case of succinic anhydride spacer. In contrast, MBS produces total abrogation of the antibody response with a high response toward the coupling agent.

Immunisation with phage-displayed variable region 2 from meningococcal PorA outer membrane protein induces bactericidal antibodies against Neisseria meningitidis.

The immunogenicity and functional activity of antibodies raised in mice against the cyclic disulphide peptide corresponding to the variable region 2 of PorA outer membrane protein from Neisseria meningitidis strain B385 (serosubtype P1.15), displayed on filamentous phage, were evaluated. The epitope, flanked either by cysteine or cysteine and three glycine residues, was expressed as a fusion to PVIII protein from M13. Immunisation of Balb/C mice with either phage generated antibody specific responses. Sera raised against the phage exposing the cyclic peptide through the three-glycine linker recognised the native protein better than those raised against the peptide with no linker. Only the phage displaying the cyclic peptide with linker was capable of inducing antibodies with bactericidal activity. These results indicate the possibility of using phage display for conformational peptide expression for immunisation to elicit functional antibody responses.

Immunological evaluation of Escherichia coli-derived hepatitis C virus second envelope protein (E2) variants.

Two variants of the hepatitis C virus (HCV) E2 envelope protein, lacking the C-terminal domain and comprising amino acids 458-650 (E2A) and 382-605 (E2C), respectively, were efficiently produced in BL21 (DE3) Escherichia coli cells. E2A and E2C were used to immunize mice. The E2C variant induced the maximal mean antibody titer. Anti-E2C mouse sera reacted mainly with E2 synthetic peptides covering the 70 amino acid N-terminal region of the E2 protein. Moreover, a panel of anti-HCV positive human sera recognized only the E2C protein (28.2%) and the synthetic peptide covering the HVR-1 of the E2 protein (23.1%). These data indicate the existence of an immunologically relevant region in the HVR-1 of the HCV E2 protein.

Anti-PorA antibodies elicited by immunization with peptides conjugated to P64k.

To increase the humoral immune response against two cyclic synthetic peptides, derived from variable regions within the outer membrane meningococcal protein PorA (subtypes 19 and 15), we conjugated the peptides to P64k, a novel carrier protein from the same bacterium expressed in Escherichia coli. In addition, one of these peptides was restricted to a linear conformation before it was chemically coupled to the carrier. The conjugates were administered to mice in a three-dose immunization schedule, resulting in a potent anti-peptide immune response, which suggested that chemical conjugation to this carrier provided T-cell help. Antisera directed to the three conjugates reacted with Neisseria meningitidis outer membrane PorA upon immunoblot analysis. Moreover, in two out of three conjugates, the anti-peptide sera reacted with native meningococcal outer membrane vesicles in ELISA.

A Limulus antilipopolysaccharide factor-derived peptide exhibits a new immunological activity with potential applicability in infectious diseases.

Previous studies have shown that cyclic peptides corresponding to residues 35 to 52 of the Limulus antilipopolysaccharide (anti-LPS) factor (LALF) bind and neutralize LPS-mediated in vitro and in vivo activities. Therapeutic approaches based on agents which bind and neutralize LPS activities are particularly attractive because these substances directly block the primary stimulus for the entire proinflammatory cytokine cascade. Here we describe new activities of the LALF(31-52) peptide, other than its LPS binding ability. Surprisingly, supernatants from human mononuclear cells stimulated with the LALF peptide are able to induce in vitro antiviral effects on the Hep-2 cell line mediated by gamma interferon (IFN-gamma) and IFN-alpha. Analysis of the effect of LALF(31-52) on tumor necrosis factor (TNF) and nitric oxide (NO) production by LPS-stimulated peritoneal macrophages revealed that a pretreatment with the peptide decreased LPS-induced TNF production but did not affect NO generation. This indicates that the LALF peptide modifies the LPS-induced response. In a model in mice with peritoneal fulminating sepsis, LALF(31-52) protected the mice when administered prophylactically, and this effect is related to reduced systemic TNF-alpha levels. This study demonstrates, for the first time, the anti-inflammatory properties of the LALF-derived peptide. These properties widen the spectrum of the therapeutic potential for this LALF-derived peptide and the molecules derived from it. These agents may be useful in the prophylaxis and therapy of viral and bacterial infectious diseases, as well as for septic shock.

Immunogenicity comparison of a multi-antigenic peptide bearing V3 sequences of the human immunodeficiency virus type 1 with TAB9 protein in mice.

The multiple antigenic peptide system (MAP) has been proposed as a novel and valuable approach for eliciting antibodies for peptides and developing synthetic vaccines. Multi-epitope polypeptides (MEP) have also been developed as an alternative to the recombinant approach for vaccines. The V3 loop from the HIV type 1 (HIV-1) external glycoprotein (gp120) contains the principal neutralization domain (PND). Antibodies against this region neutralize HIV-1 in vitro and in vivo. In this work, a novel presentation of di-epitope MAP was synthesized. A monomeric MAP carrying two identical JY1 V3 sequences as B-cell epitopes and the 830-843 region of tetanus toxoid as a T-helper cell epitope was synthesized. This basic structure was covalently linked to produce a four-JY1-branched homodimer (JY1-MAP4). Additionally, six different monomeric MAPs, bearing four copies of V3 from isolates LR150, JY1, RF, MN, BRVA and IIIB, were synthesized. These monomers were conveniently linked among themselves to produce homodimeric and heterodimeric MAPs of eight V3 branches (V3-MAP8). JY1-MAP8 elicited higher antibody titers in Balb/c mice than JY1-MAP4. The immunogenicity of two different, hexavalent V3-MAP8 mixtures and the MEP TAB9, which tandems the same six V3 sequences in a single molecule, were compared. The antibody response against the mixtures of the heterodimeric MAP showed a wider recognition pattern of the V3 region, while the homodimeric cocktail showed an intermediate pattern. Antibodies elicited by TAB9 recognized only the JY1, LR150 peptides. These results emphasize the influence of V3 epitope presentation upon the characteristics of the antibody response generated.

Effect of the position of a basic amino acid on C-terminal rearrangement of protonated peptides upon collision-induced dissociation.

Internal rearrangement involving the loss of the C-terminal amino acid residue upon collision-induced dissociation (CID) or metastable decomposition was studied for protonated peptides. To investigate the structural characteristics of peptides responsible for this rearrangement, a series of synthetic peptides were prepared and subjected to B/E-linked scan or tandem mass spectrometric analyses using a four-sector instrument. The results showed that the position of a basic amino acid in the peptide sequence and its basicity have a significant influence on the rearrangement. Arginine (Arg) located at the n-1 position facilitates the rearrangement with about twice as many rearrangement ions as is observed for the other Arg-containing peptides. This can be attributed to the interaction of a positively charged guanidino group of Arg with its own carbonyl group via a salt bridge which is tightly formed in vacuo between a guanidino and carboxylate groups, the mechanism of which is analogous to that previously proposed for the formation of similar rearrangement ions observed in the spectra of metal-cationized peptides. This association would result in the facile attack of the C-terminal hydroxyl group on the penultimate carbonyl group, leading to the rearrangement. In addition, the rearrangement ion was observed both in metastable decomposition and high-energy CID spectra obtained by B/E-linked scan analyses without or with gas, respectively, but in a sequence dependent manner.