Antigenized antibodies expressing Vβ8.2 TCR peptides immunize against rat experimental allergic encephalomyelitis

Background  

Immunity against the T cell receptor (TCR) is considered to play a central role in the regulation of experimental allergic encephalomyelitis (EAE), a model system of autoimmune disease characterized by a restricted usage of TCR genes. Methods of specific vaccination against the TCR of pathogenetic T cells have included attenuated T cells and synthetic peptides from the sequence of the TCR. These approaches have led to the concept that anti-idiotypic immunity against antigenic sites of the TCR, which are a key regulatory element in this disease.     

Utilization of multiple phage display libraries for the identification of dissimilar peptide motifs that bind to a B7-1 monoclonal antibody

Summary Seven random peptide libraries (two displaying linear peptides and five displaying cysteine-constrained peptides) were constructed as gene III fusion proteins of the bacteriophage fd-tet. These libraries were used to screen a blocking monoclonal antibody raised against B7-1 (CD80), a human cell surface antigen that binds two T cell receptors, CD28 and CTLA-4. After three rounds of screening against the immobilized antibody, 1000-fold enrichment was observed in libraries displaying both linear and cysteineconstrained peptides. DNA sequencing of the enriched phage revealed two distinct consensus sequences: HXG(A/Y)XH and DVCXXGGPGC. Phage expressing these consensus sequences bound to L307.4 but not to an isotype matched antibody, indicating that binding was antibody specific. Synthetic peptides corresponding to both motifs inhibited phage binding to L307.4, indicating that the gene III protein is not required for peptide binding. In addition, the cyclized forms of synthetic peptides containing the DVCXXGGPGC motif were capable of inhibiting L307.4 binding to soluble B7-1/Fc fusion. Moreover, phage expressing only the HXG(A/Y)XH consensus sequence were inhibited from binding to L307.4 by the presence of chelating agents. These results indicate that the framework within which the peptide is presented on the surface of the phage may allow the identification of unique peptide motifs with distinct binding characteristics. These peptide motifs could be used for the design of peptidomimetics with therapeutic applications if they inhibit the binding of B7-1 to its T cell receptors.     

Detection of Cancer Cell Death Mediated by a Synthetic Granzyme B-like Peptide Fluorescent Conjugate and the same Peptide Binding in Bacteria

Granzyme-mediated apoptosis, supported by pore-forming perforin, plays an important role in CD8+ T lymphocytes (CTL)-dependent cellular immunity protection against both cancer and viral infection. Quantitative and qualitative problems with CTL are potential contributing factors to disease progression. The feasibility of developing CTL-independent cellular immunity is desired but must first overcome the barrier of CTL-independent target cell recognition. Granzyme B with its strong pro-apoptotic activity in many different target cells is investigated for use in the CTL-independent cellular immunity approach, and granzyme B or its bioactive peptides without the enzymatic activity are more desirable for use. Native granzyme B with enzymatic activity is usually investigated in cancer cells for its mediation of apoptosis by detection of DNA fragmentation. Detection of cell death mediated by such peptides in cancer cells is needed to demonstrate the potential therapeutic purposes. We show with never-before-seen microscopic images using fluorescence microscopy that a synthetic granzyme B-like peptide fluorescent conjugate (GP1R) can: 1) mediate cell death of different cancer cells via membrane extrusion, 2) bind to constitutively expressed binding targets in different cancer cells and bacteria, and 3) promote bacterial phagocytosis. The putative binding targets may serve as a universal pathologic biomarker detectable by GP1R. Our data taken together demonstrate the potential applications of GP1R for use in CTL-independent target cell recognition and target cell death induction. It may lead to development of rapid targeted detection and new treatment of cancer, viral and bacterial infections. The new treatment may show mutual benefits for two or more diseases.     

Epidemic spreading on networks with vaccination

In this paper, a new susceptible-infected-susceptible (SIS) model on complex networks with imperfect vaccination is proposed. Two types of epidemic spreading patterns (the recovered individuals have or have not immunity) on scale-free networks are discussed. Both theoretical and numerical analyses are presented. The epidemic thresholds related to the vaccination rate, the vaccination-invalid rate and the vaccination success rate on scale-free networks are demonstrated, showing different results from the reported observations. This reveals that whether or not the epidemic can spread over a network under vaccination control is determined not only by the network structure but also by the medicine's effective duration. Moreover, for a given infective rate, the proportion of individuals to vaccinate can be calculated theoretically for the case that the recovered nodes have immunity. Finally, simulated results are presented to show how to control the disease prevalence.     

Tumor-infiltrating effector cells of alpha-galactosylceramide-induced antitumor immunity in metastatic liver tumor

BACKGROUND: alpha-Galactosylceramide (alpha-GalCer) can be presented by CD1d molecules of antigen-presenting cells, and is known to induce a potent NKT cell-dependent cytotoxic response against tumor cells. However, the main effector cells in alpha-GalCer-induced antitumor immunity are still controversial. METHODS: In order to elucidate the cell phenotype that plays the most important role in alpha-GalCer-induced antitumor immunity, we purified and analyzed tumor-infiltrating leukocytes (TILs) from liver metastatic nodules of a colon cancer cell line (Colon26), comparing alpha-GalCer- and control vehicle-treated mice. Flow cytometry was performed to analyze cell phenotype in TILs and IFN-gamma ELISA was performed to detect antigen-specific immune response. RESULTS: Flow cytometry analysis showed a significantly higher infiltration of NK cells (DX5+, T cell receptor alphabeta (TCR)-) into tumors in alpha-GalCer-treated mice compared to vehicle-treated mice. The DX5+TCR+ cell population was not significantly different between these two groups, indicating that these cells were not the main effector cells. Interestingly, the CD8+ T cell population was increased in TILs of alpha-GalCer-treated mice, and the activation level of these cells based on CD69 expression was higher than that in vehicle-treated mice. Moreover, the number of tumor-infiltrating dendritic cells (DCs) was increased in alpha-GalCer-treated mice. IFN-gamma ELISA showed stronger antigen-specific response in TILs from alpha-GalCer-treated mice compared to those from vehicle-treated mice, although the difference between these two groups was not significant. CONCLUSIONS: In alpha-GalCer-induced antitumor immunity, NK cells seem to be some of the main effector cells and both CD8+ T cells and DCs, which are related to acquired immunity, might also play important roles in this antitumor immune response. These results suggest that alpha-GalCer has a multifunctional role in modulation of the immune response.     

Effective vaccination strategies for realistic social networks

We consider the effectiveness of targeted vaccination at preventing the spread of infectious disease in a realistic social network. We compare vaccination strategies based on no information (random vaccination) to complete information (PageRank) about the network. The most effective strategy we find is to vaccinate those people with the most unvaccinated contacts. However, this strategy requires considerable information and computational effort which may not be practical. The next best strategies vaccinate people with many contacts who in turn have few contacts.     

A novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens

The heat shock protein, Hsp70, has been shown to play an important role in tumour immunity. Vaccination with Hsp70-peptide complexes (Hsp70-PCs), isolated from autologous tumour cells, can induce protective immune responses. We have developed a novel method to identify synthetic mimic peptides of Hsp70-PCs and to test their ability to activate T-cells. Peptides (referred to as "recognisers") that bind to Hsp70-PCs from the human breast carcinoma cell line, MDA-MB-231, were identified by bio-panning a random peptide M13 phage display library. Synthetic recogniser peptides were subsequently used as bait in a reverse bio-panning experiment to identify potential Hsp70-PC mimic peptides. The ability of the recogniser and mimic peptides to prime human lymphocyte responses against tumour cell antigens was tested by stimulating lymphocytes with autologous peptide-loaded monocyte-derived dendritic cells (DCs). Priming and subsequent stimulation with either the recogniser or mimic peptide resulted in interferon-γ (IFN-γ) secretion by the lymphocytes. Furthermore, DCs loaded with Hsp70, Hsp70-PC or the recogniser or the mimic peptide primed the lymphocytes to respond to soluble extracts from breast cells. These results highlight the potential application of synthetic peptide-mimics of Hsp70-PCs, as modulators of the immune response against tumours.     

Correlation between MRI and clinico-pathological manifestations in Lewis rats protected from experimental allergic encephalomyelitis by acylated synthetic peptide of myelin basic protein.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system which constitutes an accepted animal model for multiple sclerosis (MS). The disease can take an acute or chronic form depending on the injection route, animal strain and nature of the disease-inducing antigen administered. The neuroinflammation associated with the acute form can be detected with T2-weighted, T1-weighted and diffusion MRI, and blood-brain barrier changes can be investigated with Gd-DTPA-enhanced T1-weighted imaging, similar to that of MS patients. A synthetic peptide of myelin basic protein (MBP) encephalitogenic for the Lewis rat (MBP 68-86) was acylated by the attachment of a palmitoyl residue (PAL68-86), and was shown to confer almost complete protection against EAE, when administered to rats before and after an encephalitogenic challenge. In this study, treatment of Lewis rats with PAL68-86 prevented the appearance of clinical signs (p < 0.0001) after challenge with the native peptide (p68-86) in complete Freund's adjuvant (CFA), and reduced considerably the MRI and histopathological signs of the disease (p < 0.0001). Measurement of the gadolinium leakage due to neuroinflammation revealed a significant decrease in permeability from 4.09 +/- 2.1 to 2.95 +/- 1.79% pixels > mean + 2 SD (p = 0.011). Therefore, quantitative MRI measurements correlate very well with the reduced cellular infiltration in the CNS and the absence of clinical signs in the EAE-protected animal.     

Paratuberculosis control: a review with a focus on vaccination

Mycobacterium avium subsp. paratuberculosis (MAP) infection causes in ruminants a regional chronic enteritis that is increasingly being recognized as a significant problem affecting animal health, farming and the food industry due to the high prevalence of the disease and to recent research data strengthening the link between the pathogen and human inflammatory bowel disease (IBD). Control of the infection through hygiene-management measures and test and culling of positive animals has to date not produced the expected results and thus a new focus on vaccination against this pathogen is necessary. This review summarizes all vaccination studies of cattle, sheep or goats reporting production, epidemiological or pathogenetic effects of vaccination published before January 2010 and that provide data amenable to statistical analyses. The meta analysis run on the selected data, allowed us to conclude that most studies included in this review reported that vaccination against MAP is a valuable tool in reducing microbial contamination risks of this pathogen and reducing or delaying production losses and pathogenetic effects but also that it did not fully prevent infection. However, the majority of MAP vaccines were very similar and rudimentary and thus there is room for improvement in vaccine types and formulations.     

Tumor-associated antigens identified by mRNA expression profiling as tumor rejection epitopes

Thirteen H-2b-binding peptides derived from six potentially overexpressed proteins in p53-/- thymoma (SM7) cells were studied for immunogenecity and vaccine-induced prevention of tumor growth in mice inoculated with SM7 tumor cells. Six of the peptides generated specific CTL responses after immunization, but only two of these peptides (RAD23-31 and RAD24-31) were capable of generating a weak vaccination-induced protection against adoptive tumor growth. SM7 inoculated mice treated with a blocking antibody against the inhibitory T cell signal transducing molecule CTLA4 appeared to delay tumor take, suggesting that SM7 thymoma cells are recognized by the adaptive immune system of the host. However, prophylactic vaccination with RAD23-31 and RAD24-31 peptides combined with anti-CTLA4 Ab treatment and did not improve tumor resistance. Our data would indicate that vaccination with immunogenic peptides derived from potentially overexpressed tumor proteins, as identified by mRNA expression profiling of p53-/- thymoma cells, at best results in a weak tumor protection thus questioning this way of detection of new tumor rejection epitopes.     

Neuroprotective effects of edaravone-administration on 6-OHDA-treated dopaminergic neurons

Background

A recent human clinical trial of an Alzheimer's disease (AD) vaccine using amyloid beta (Aβ) 1–42 plus QS-21 adjuvant produced some positive results, but was halted due to meningoencephalitis in some participants. The development of a vaccine with mutant Aβ peptides that avoids the use of an adjuvant may result in an effective and safer human vaccine.

Results

All peptides tested showed high antibody responses, were long-lasting, and demonstrated good memory response. Epitope mapping indicated that peptide mutation did not lead to epitope switching. Mutant peptides induced different inflammation responses as evidenced by cytokine profiles. Ig isotyping indicated that adjuvant-free vaccination with peptides drove an adequate Th2 response. All anti-sera from vaccinated mice cross-reacted with human Aβ in APP/PS1 transgenic mouse brain tissue.

Conclusion

Our study demonstrated that an adjuvant-free vaccine with different Aβ peptides can be an effective and safe vaccination approach against AD. This study represents the first report of adjuvant-free vaccines utilizing Aβ peptides carrying diverse mutations in the T-cell epitope. These largely positive results provide encouragement for the future of the development of human vaccinations for AD.     

Peptide antagonists of superantigen toxins

Superantigens produced by Staphylococcus aureus and Streptococcus pyogenes are among the most lethal of toxins. Toxins in this large family trigger an excessive cellular immune response leading to toxic shock. Superantigens are secreted by the bacteria as diverse natural mixtures, a complexity that demands development of broad-spectrum countermeasures. We used a rational approach to design short peptides with homology to various domains in a typical superantigen (staphylococcal enterotoxin B) and screened each peptide for its ability to antagonize, in human peripheral blood mononuclear cells, superantigen-mediated induction of the genes encoding T helper 1 cytokines that mediate shock: interleukin-2, interferon-gamma and tumor necrosis factor. A dodecamer peptide proved a potent antagonist against widely different superantigens. This peptide protected mice from killing by superantigens and it was able to rescue mice undergoing toxic shock. The antagonist peptide shows homology to a beta-strand-hinge-alpha-helix domain that is structurally conserved among superantigens, yet currently of unknown function and remote from the binding sites for the known ligands essential for T cell activation, the major histocompatibility complex class II molecule and T cell receptor. The antagonist activity of this peptide thus identifies a novel domain in superantigens that is critical for their toxic action. The antagonist peptide provides a new tool for understanding the mechanism of excessive human immune response activation by superantigens that occurs during toxic shock and for identification of a novel target ligand that may interact with this superantigen domain.     

Improved generation of anti-tumor immunity by antigen dose limitation

Background  

The malignant cells of cutaneous T cell lymphoma (CTCL) display immunogenic peptides derived from the clonal T cell receptor (TCR) providing an attractive model for refinement of anti-tumor immunization methodology. To produce a clinically meaningful anti-tumor response, induction of cytotoxic anti-CTCL cells must be maximized while suppressive T regulatory cells (Treg) should be minimized. We have demonstrated that engulfment of apoptotic CTCL cells by dendritic cells (DC) can lead to either CD8 anti-CTCL responses or immunosuppressive Treg induction. Treg generation is favored when the number of apoptotic cells available for ingestion is high.     

The Influence of T Cell Development on Pathogen Specificity and Autoreactivity

T cells orchestrate adaptive immune responses upon activation. T cell activation requires sufficiently strong binding of T cell receptors on their surface to short peptides derived from foreign proteins bound to protein products of the major histocompatibility (MHC) gene products, which are displayed on the surface of antigen presenting cells. T?cells can also interact with peptide-MHC complexes, where the peptide is derived from host (self) proteins. A diverse repertoire of relatively self-tolerant T cell receptors is selected in the thymus. We study a model, computationally and analytically, to describe how thymic selection shapes the repertoire of T cell receptors, such that T cell receptor recognition of pathogenic peptides is both specific and degenerate. We also discuss the escape probability of autoimmune T cells from the thymus.     

Active inflammation increases the heterogeneity of MRI texture in mice with relapsing experimental allergic encephalomyelitis

Inflammation modulates tissue damage in relapsing-remitting multiple sclerosis (MS) both acutely and chronically, but its severity is difficult to evaluate with conventional MRI analysis. In mice with experimental allergic encephalomyelitis (EAE, a model of MS), we administered ultra small particles of iron oxide to track macrophage-mediated inflammation during the onset (relapse) and recovery (remission) of disease activity using high field MRI. We performed MRI texture analysis, a sensitive measure of tissue regularity, and T2 assessment both in EAE lesions and the control tissue, and measured spinal cord volume. We found that inflammation was 3 times more remarkable at onset than at recovery of EAE in histology yet demyelination appeared similar across animals and disease course. In MRI, lesion texture was more heterogeneous; T2 was lower; and spinal cord volume was greater in EAE than in controls, but only MRI texture was worse at relapse than at remission of EAE. Moreover, MRI texture correlated with spinal cord volume and tended to correlate with the extent of disability in EAE. While subject to further confirmation, our findings may suggest the sensitivity of MRI texture analysis for accessing inflammation.     

Use of peptide for selective and sensitive detection of an Anthrax biomarker via peptide recognition and surface‐enhanced Raman scattering

A short 16‐amino acid peptide has been used in place of an antibody to selectively detect the specific Anthrax biomarker, protective antigen (PA), using surface‐enhanced Raman scattering (SERS). Peptides are more stable than antibodies under various biological conditions and are easily synthesized for a specific target. A peptide that has high affinity to PA was conjugated onto gold nanoparticles along with a Raman reporter and then incubated in various concentrations of PA. Parallel studies in which the peptide sequence was replaced with an antibody were performed to compare the performance of the two methodologies. Both the peptide and antibody functionalized nanoparticles were able to specifically detect PA concentrations down to 6.1 fM . These results demonstrate that these short, robust peptides can be used in the place of traditional antibodies to specifically recognize target biomarkers in the field for the potential diagnosis of disease. Copyright © 2010 John Wiley & Sons, Ltd.     

Prospects for control of emerging infectious diseases with plasmid DNA vaccines

Experiments almost 20 years ago demonstrated that injections of a sequence of DNA encoding part of a pathogen could stimulate immunity. It was soon realized that "DNA vaccination" had numerous potential advantages over conventional vaccine approaches including inherent safety and a more rapid production time. These and other attributes make DNA vaccines ideal for development against emerging pathogens. Recent advances in optimizing various aspects of DNA vaccination have accelerated this approach from concept to reality in contemporary human trials. Although not yet licensed for human use, several DNA vaccines have now been approved for animal health indications. The rapid manufacturing capabilities of DNA vaccines may be particularly important for emerging infectious diseases including the current novel H1N1 Influenza A pandemic, where pre-existing immunity is limited. Because of recent advances in DNA vaccination, this approach has the potential to be a powerful new weapon in protecting against emerging and potentially pandemic human pathogens.     

Generating neutralizing antibodies,Th1 response and MHC non restricted immunogenicity of HIV-I <Emphasis Type="Underline">env</Emphasis> and <Emphasis Type="Underline">gag</Emphasis>peptides in liposomes and ISCOMs with in-built adjuvanticity

For enhancing immunogenicity and develop vaccine strategies using peptide based constructs against HIV-1, a chimeric peptide containing V3 loop and transmembrane sequence of gp41 with two glycine motifs as spacer was constructed. The V3-gp41, gp41 peptide and p17 and p24 peptides separately or in a cocktail were entrapped with or without MA729 as an immunoadjuvant in liposomes or ISCOMs. The immunogenicity, antigen induced T-cell proliferation and cytokine profiles of various formulations were studied in four different inbred strains of mice of H-2^d, H-2^b, H-2^k and H-2^q haplotypes, keeping alum as a control adjuvant. Both liposomes and ISCOM preparations elicited high titer and long lasting antibody response (60 days and above). When compared to the alum formulation, the liposomes co-entrapped with MA729 produced high antibody levels, comparable with that induced by ISCOMs. Peptide in alum, liposomes and ISCOMs enhanced both antigen specific IgG2a and IgG2b isotypes and high T-cell stimulation index. Peptide formulations also induced antibodies with high affinity and in vitro neutralizated the formation of HIV-1 syncytia. T-cell supernatants contained high levels of IFN-γ and IL-2. Thus formulation in these adjuvants induced a predominant Th1 like response with MA729 as a versatile novel delivery vehicle for stimulating the appropriate arm of the immune response that can selectively modulate MHC class I or MHC class II response. The above peptide can be of wide vaccination interest as a means to improve immune responses to several other HIV-1 antigens and may serve as candidates for vaccine development.     

Synthesis and Characterization of Acylated Polycaprolactone (PCL) Nanospheres and Investigation of Their Influence on Aggregation of Amyloid Proteins

Acylated polycaprolactone (PCL) nanospheres were fabricated and employed to interact with amyloid-β-(25–35) peptides (Aβ_25–35), "peptide 11 of the 40 peptide full amyloid-β". The nanospheres were characterized by scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, and dynamiclightscattering (DLS), all of which confirmed that the acylated polycaprolactone (Ac-PCL) nanospheres were successfully fabricated. The effect of the nanospheres on the aggregation of Aβ_25–35 peptides was investigated by thioflavin T fluorescence measurements. The result showed that, without nanospheres, the Aβ_25–35 peptides aggregated gradually from monomers and oligomers to long fibrils with increasing incubation time. In comparison, the nanospheres were effective in interfering with fibrillogenesis and aggregation of amyloid-β. We suggest this study may contribute to the development of new therapeutic strategies against amyloid-related disorders.     

Identification of peptide sequences that bind the Thomsen-Friedenreich cancer-associated glycoantigen from bacteriophage peptide display libraries

Summary The goal of this study was to determine if polypeptides that bind specifically to the carcinoma-associated Thomsen-Friedenreich (T) antigen could be isolated from a random peptide bacteriophage display library. T antigen is a carbohydrate antigen that is exposed and immunoreactive on the surfaces of most primary carcinomas and their metastases, while it is masked on normal cells. Tumor-specific surface carbohydrates are often used as markers of cell differentiation and play a role in cell aggregation, which is an important step in the metastatic process. Therefore, peptides that bind and mask T antigen may yield useful carbohydrate-specific probes and provide insight into carbohydrate-mediated tumor-cell aggregation. A 15-amino acid random peptide bacteriophage display library was screened for polypeptides that exhibited high specificity to two glycoproteins which display T antigen on their surfaces. The results suggest that synthetic peptides identified from the bacteriophage display library have high affinities (K_d 1 M) and specificities for proteins and human tumor cells which present T antigen. Thus, random bacteriophage peptide display libraries may be a rich source of sequences that bind to carbohydrate antigen structures.