Behjatolah Monzavi-Karbassi

Peptide mimotopes as surrogate antigens of carbohydrates in vaccine discovery

Carbohydrate antigens are immune targets associated with a variety of pathogens and tumor cells. Unfortunately, most carbohydrates are intrinsically T cell-independent antigens, which diminishes their efficacy as immunogens. The conversion of carbohydrate epitopes to peptide mimotopes is one means to overcome the T cell-independent nature of carbohydrate antigens because peptides have an absolute requirement for T cells. Although such conversion has great potential for the development of veterinarian and human vaccines, there are issues related to the use of peptide-based immunogens as functional surrogates. Some of these issues are fundamental, pertaining to how mimicry comes about at the molecular level, and some are application oriented, directed at elucidating important immunological mechanisms. In this article the potential and caveats of this technology regarding its application in vaccine discovery are analyzed.

Chondroitin sulfates play a major role in breast cancer metastasis: a role for CSPG4 and CHST11 gene expression in forming surface P-selectin ligands in aggressive breast cancer cells

IntroductionWe have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread.MethodsQuantitative real-time PCR (qRT-PCR) and flow cytometry assays were used to detect the expression of genes involved in the sulfation and presentation of chondroitin in several human breast cancer cell lines. Transient transfection of the human breast cancer cell line MDA-MB-231 with the siRNAs for carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) and chondroitin sulfate proteoglycan 4 (CSPG4 ) was used to investigate the involvement of these genes in expression of surface P-selectin ligands. The expression of CSPG4 and CHST11 in 15 primary invasive breast cancer clinical specimens was assessed by qRT-PCR. The role of CS-GAGs in metastasis was tested using the 4T1 murine mammary cell line (10 mice per group).ResultsThe CHST11 gene was highly expressed in aggressive breast cancer cells but significantly less so in less aggressive breast cancer cell lines. A positive correlation was observed between the expression levels of CHST11 and P-selectin binding to cells (P < 0.0001). Blocking the expression of CHST11 with siRNA inhibited CS-A expression and P-selectin binding to MDA-MB-231 cells. The carrier proteoglycan CSPG4 was highly expressed on the aggressive breast cancer cell lines and contributed to the P-selectin binding and CS-A expression. In addition, CSPG4 and CHST11 were over-expressed in tumor-containing clinical tissue specimens compared with normal tissues. Enzymatic removal of tumor-cell surface CS-GAGs significantly inhibited lung colonization of the 4T1 murine mammary cell line (P = 0.0002).ConclusionsCell surface P-selectin binding depends on CHST11 gene expression. CSPG4 serves as a P-selectin ligand through its CS chain and participates in P-selectin binding to the highly metastatic breast cancer cells. Removal of CS-GAGs greatly reduces metastatic lung colonization by 4T1 cells. The data strongly indicate that CS-GAGs and their biosynthetic pathways are promising targets for the development of anti-metastatic therapies.

Cutting Edge: DNA Immunization with Minigenes of Carbohydrate Mimotopes Induce Functional Anti-Carbohydrate Antibody Response

To date, the generation of anti-carbohydrate Th1 immune responses, which would be useful for both tumor immunotherapy as well as in pathogen vaccine strategies, has been elusive. To augment Th1 immune responses to carbohydrate Ags, we describe results of DNA vaccination studies in mice using plasmids encoding designed peptide mimotopes (minigenes) of the neolactoseries Ag Lewis Y (LeY). In contrast to LeY immunization, immunization with mimotope-encoded plasmids induced LeY cross-reactive IgG2a Abs. Minigene immunization primed for a LeY-specific response that is rapidly activated upon encounter with nominal Ag upon subsequent boost. The resulting IgG2a response mediated complement-dependent cytotoxicity of a LeY-expressing human tumor cell line in the presence of human complement. These studies establish that peptide mimotopes of carbohydrate Ags encoded as DNA plasmids are novel immunogens providing a means to manipulate carbohydrate cross-reactive Th1 responses.

Chondroitin sulfate glycosaminoglycans as major P‐selectin ligands on metastatic breast cancer cell lines

The metastatic breast cancer cell line, 4T1, abundantly expresses the oligosaccharide sialylated Lewis x (sLex). SLex oligosaccharide on tumor cells can be recognized by E‐ and P‐selectin, contributing to tumor metastatic process. We observed that both selectins reacted with this cell line. However, contrary to the E‐selectin reactivity, which was sLex dependent, P‐selectin reactivity with this cell line was sLex‐independent. The sLex‐Neg variant of the 4T1 cell line with markedly diminished expression of sLex and lack of sLea, provided a unique opportunity to characterize P‐selectin ligands and their contribution to metastasis in the absence of overlapping selectin ligands and E‐selectin binding. We observed that P‐selectin binding was Ca2+‐independent and sulfation‐dependent. We found that P‐selectin reacted primarily with cell surface chondroitin sulfate (CS) proteoglycans, which were abundantly and stably expressed on the surface of the 4T1 cell line. P‐selectin binding to the 4T1 cells was inhibited by heparin and CS glycosaminoglycans (GAGs). Moreover, Heparin administration significantly inhibited experimental lung metastasis. In addition, the data suggest that surface CS GAG chains were involved in P‐selectin mediated adhesion of the 4T1 cells to murine platelets and human umbilical vein endothelial cells. The data suggest that CS GAGs are also the major P‐selectin‐reactive ligands on the surface of human MDA‐MET cells. The results warrant conducting clinical studies on the involvement of cell surface CS chains in breast cancer metastasis and evaluation of various CS types and their biosynthetic pathways as target for development of treatment strategies for antimetastatic therapy of this disease.

Peptide mimotopes of carbohydrate antigens

Carbohydrate structures have been identified as significant antigens for bacterial, viral, and fungal pathogens as well as targets on human tumor cells. Many of these antigens are poorly immunogenic in humans, requiring extensive adjuvant sublimation. Although conjugate carbohydrate vaccines appear promising, there are limitations of using carbohydrate formulations. An alternative approach is to use surrogate antigens for some carbohydrates. We are developing peptides that mimic carbohydrates which might be further manipulated to induce responses that target biologically important carbohydrates expressed on pathogens and on tumor cells. We have shown that peptide mimotopes of carbohydrates induce immune responses to carbohydrate structures with in vivo and vitro functionality. Model systems include the Neisseria group C meningococcal polysaccharide; the histo-blood group-related antigens expressed on tumor cells; and mannose, sialyl, and histo-blood group-related carbohydrate epitopes expressed on human immunodeficiency virus.

Priming characteristics of peptide mimotopes of carbohydrate antigens.

Immunization with peptide mimetics of carbohydrate antigens can induce functional carbohydrate-reactive antibodies. Here, we examine the immune characteristics of alternative approaches in prime and boost strategies using glycosylated HIV-1 envelope protein and model tumor associated carbohydrate antigens. Our results indicate that peptide mimotopes either in a DNA or carrier-conjugated format can induce comparable levels of IgM and IgG. Carbohydrate boosting of peptide-primed animals does not affect end-point titer, however, boosting mediates a stable long lasting carbohydrate reactive IgM response, not achievable by carbohydrate immunization alone. Boosting with carbohydrate in animals primed with DNA- or peptide-conjugate, facilitates the induction of detectable IgG with a dominant IgG2a isotype. Immunization with HIV-1 envelope glycoprotein of peptide-primed animals induces different IgG isotype profiles with a dominant IgG1 antibody. We observed that HIV-1 envelope glycoprotein immunization of peptide primed mice induces a cross-reactive cellular response, as detected by cytokine secretion, which lends to IFN-gamma production upon splenocyte stimulation and CTL activity against recombinant vaccinia virus infected cells after in vitro stimulation. DNA immunization with mimotope, inclusion of a T-cell epitope from the HIV-1 envelope protein in the expression cassette and co-administration with IL-12 or GM-CSF encoding plasmids activate a cellular response to the HIV-1 envelope protein.

The use of a thermostable beta-glucanase gene from Clostridium thermocellum as a reporter gene in plants.

Abstract In order to take advantage of the high thermostability of its product, β-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine TR-DNA 2′ gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering.

Antigenic properties of peptide mimotopes of HIV-1-associated carbohydrate antigens

The glycan shield of the human immunodeficiency virus (HIV) envelope protein presents many potential epitopes for vaccine development. To augment immune responses to HIV, type 1 (HIV-1), envelope-associated carbohydrate antigens, we are defining peptide mimics of HIV-associated carbohydrate antigens that function as antigen mimotopes that upon immunization will induce antibodies cross-reactive with carbohydrate antigens. We have previously defined peptides with a putative sequence tract RYRY that mimic concanavalin A-binding glycans. To imitate the multivalent binding of carbohydrates, we compared the avidity of a linear (911) and cyclic peptide (D002) reactive with concanavalin A presented in a multiple antigen peptide (MAP) format. The affinity of the MAP-D002 peptide was higher than that of the peptide MAP-911, whereas the avidity of D002 peptide was lower than that of 911. Serum from mice immunized with MAP-911 had lower titer for oligomannose-9 than those elicited by MAP-D002 under the same conditions, but both immunogens elicited antibodies that can block the binding of GP120 to dendritic cells. Antibodies that bind to the studied MAPs were found in a preparation of normal human immunoglobulin for intravenous use. Those that were purified on 911 bound back to 911 and D002, whereas anti-D002 antibodies were specific only for D002. Human antibodies reactive with both mimotopes and with a mannosyl preparation were observed to bind to envelope protein. These results suggested the potential to fine-tune the antibody response to carbohydrate antigens by modifying structural features of peptide mimotope-based immunogens.

A mimic of tumor rejection antigen-associated carbohydrates mediates an antitumor cellular response.

Tumor-associated carbohydrate antigens are typically perceived as inadequate targets for generating tumor-specific cellular responses. Lectin profile reactivity and crystallographic studies demonstrate that MHC class I molecules can present to the immune system posttranslationally modified cytosolic peptides carrying O-β-linked N-acetylglucosamine (GlcNAc). Here we report that a peptide surrogate of GlcNAc can facilitate an in vivo tumor-specific cellular response to established Meth A tumors that display native O-GlcNAc glycoproteins on the tumor cell surface. Peptide immunization of tumor-bearing mice had a moderate effect on tumor regression. Inclusion of interleukin 12 in the immunization regimen stimulated complete elimination of tumor cells in all of the mice tested, whereas interleukin 12 administration alone afforded no tumor growth inhibition. Adoptive transfer of immune T cells into tumor-bearing nude mice indicates a role for CD8+ T cells in tumor regression. This work postulates that peptide mimetics of glycosylated tumor rejection antigens might be further developed for immune therapy of cancer.

Strategies in cancer vaccines development

The recent definition of tumour-specific immunity in cancer patients and the identification of tumour-associated antigens have generated renewed enthusiasm for the application of immune-based therapies for the treatment of malignancies. Recent developments in cancer vaccines have also been based on an improved understanding of the cellular interactions required to induce a specific anti-tumour immune response. Consequently, a number of cancer vaccines have entered clinical trials. Targeting broad-spectrum tumour-associated antigens has emerged as a strategy to lower the risk of tumour escape due to the loss of specific nominal antigen. Amongst the most challenging of tumour-associated antigens to which to target in active specific immunotherapy applications are carbohydrate antigens. As carbohydrates are intrinsically T-cell-independent antigens, more novel approaches are perhaps needed to drive specific-T-cell-dependent immune responses to carbohydrate antigens. In this context peptide mimetics of core structures of tumour-associated carbohydrate antigens might be developed to augment immune responses to these broad-spectrum antigens.