Fucosylated lactosamines participate in adhesion of HIV-1 envelope glycoprotein to dendritic cells.

Carbohydrates expressed on HIV-1 gp160 are purported to bind to several receptor types that affect virus pathophysiology. Here, we define a potential role for fucosylated glycans involved in the adhesion of cells expressing anchored HIV-1 glycoprotein or HIV virions to human dendritic cells (DCs). We observe that a monoclonal antibody (FH6), with reactivity toward an extended dimeric form of a fucosyl lactosamine, binds to gp120 transfectants, blocking adhesion of these cells and virus particles to human DCs. We observe that serum antibodies induced by peptide mimetic of fucosylated carbohydrate core structures emulate the monoclonal antibody reactivity pattern, showing enhanced reactivity to HIV-1 envelope-expressing cell line and blocking the adhesion of these cells to human DCs. These results suggest a potential role for initial adherence of virally infected cells or virions mediated by fucosylated lactosamines expressed on the envelope protein. As these carbohydrates function as adhesion molecules associated with homing and dissemination processes, such interactions may contribute to the HIV infection process.

Peptide mimotopes as prototypic templates of broad-spectrum surrogates of carbohydrate antigens.

Peptide mimetics of carbohydrate antigens can function as templates to exploit immune mechanisms to augment vaccine design strategies as they are T cell dependent antigens. In this study we evaluate a peptide mimetic (peptide 105) of the Pneumococcal capsular polysaccharide type 14 (Pn14) as a model antigen to explore differences in antigenicity and immunogenicity of peptide mimotopes. The multiple antigenic peptide (MAP) form, by ELISA, competes with native Pn14 in a concentration-dependent manner for binding to an anti-Pn14 monoclonal antibody. It was observed that peptide priming with a conjugated form (105-BSA) and boosting with Pn14 produced higher levels of Pn14-reactive IgG1, IgG2a, IgG2b and IgG3 than priming and boosting with Pn14. This serum also displayed reactivity with multiple serotypes, as assessed by ELISA. However, when compared with serum from humans immunized with the 23-valent pneumococcal vaccines, mimetic-induced mouse serum did not display a significant ability to mediate opsonophagocytic killing of pneumococci. These results suggest the feasibility of designing mimotopes to render effective humoral responses not only to a single serotype of Streptococcus pneumoniae, but to multiple serotypes at once. Such peptides would simplify currently available vaccine approaches, yet highlights the requirement of more extensive polymerization to fully emulate native antigen.

The hidden code in genomics: a tool for gene discovery.

Among new insights coming from the completion of sequencing of the human genome, reported in Nature and Science, are clues of how evolution has increased the complexity of species, and in particular how the genetic code has enabled this process. It is clear that life has not only evolved by increasing the number of genes, but also by ingeniously evolving an efficient code for expressing diversity in the building blocks (i.e. the amino acids). The rules of nucleic acid base pairing and the classification of amino acids according to hydrophobicity/hydrophilicity relationships define a binary DNA code, which determines the general biophysical characteristics of proteins. Sense and antisense strands can encode protein segments having inverted and complementary hydropathy. The underlying binary code controls association and dissociation of proteins and presumably represents a primordial code that might have emerged in the early stages of self-organizing biochemical cycles. It is the purpose of this communication to provide a perspective of the code in the context of a binary language from its primordial origin to its present day format and to propose to use this code as a genomic mining tool.

Immunization with a carbohydrate mimicking peptide augments tumor-specific cellular responses.

The metastatic potential of some tumor cells is associated with the expression of the neolactoseries antigens sialyl-Lewis x (sLex) and sialyl-Lewis a (sLea) as they are ligands for selectins. We have recently shown that peptide mimetics of these antigens can potentiate IgG2a antibodies, which are associated with a Th1-type cellular response. As L-selectin is preferentially expressed on CD4+ Th1 and CD8+ T cell populations, specific induction of these phenotypes could augment a response to L-selectin ligand-expressing tumor cells. Here we demonstrate that immunization with a multiple antigen peptide (MAP) mimetic of sugar constituents of neolactoseries antigens induces a MHC-dependent peptide-specific cellular response that triggers IFN-gamma production upon peptide stimulation, correlating with IgG2a induction. Surprisingly, T lymphocytes from peptide-immunized animals were activated in vitro by sLex, also triggering IFN-gamma production in a MHC-dependent manner. Stimulation by peptide or carbohydrate resulted in loss of L-selectin on CD4+ T cells confirming a Th1 phenotype. We also observed an enhancement in cytotoxic T lymphocyte (CTL) activity in vitro against sLex-expressing Meth A cells using effector cells from Meth A-primed/peptide-boosted animals. CTL activity was inhibited by both anti-MHC class I and anti-L-selectin antibodies. These results further support a role for L-selectin in tumor rejection along with the engagement by the TCR for most likely processed tumor-associated glycopeptides, focusing on peptide mimetics as a means to induce carbohydrate reactive cellular responses.

Directing the immune response to carbohydrate antigens.

Peptide mimetics may substitute for carbohydrate antigens in vaccine design applications. At present, the structural and immunological aspects of antigenic mimicry, which translate into immunologic mimicry, as well as the functional correlates of each, are unknown. In contrast to screening peptide display libraries, we demonstrate the feasibility of a structure-assisted vaccine design approach to identify functional mimeotopes. By using concanavalin A (ConA), as a recognition template, peptide mimetics reactive with ConA were identified. Designed peptides were observed to compete with synthetic carbohydrate probes for ConA binding, as demonstrated by enzyme-linked immunosorbent assay and isothermal titration calorimetry (ITC) analysis. ITC measurements indicate that a multivalent form of one particular mimetic binds to ConA with similar affinity as does trimannoside. Splenocytes from mimeotope-immunized mice display a peptide-specific cellular response, confirming a T-cell-dependent nature for the mimetic. As ConA binds to the Envelope protein of the human immunodeficiency virus, type 1 (HIV-1), we observed that mimeotope-induced serum also binds to HIV-1-infected cells, as assessed by flow cytometry, and could neutralize T-cell line adapted HIV-1 isolates in vitro, albeit at low titers. These studies emphasize that mimicry is based more upon functional rather than structural determinants that regulate mimeotope-induced T-dependent antibody responses to polysaccharide and emphasize that rational approaches can be employed to develop further vaccine candidates.

Exploiting molecular mimicry to broaden the immune response to carbohydrate antigens for vaccine development.

Peptide mimetics of carbohydrates represent an alternative approach to induce anti-carbohydrate responses. Depending on their formulation, peptide mimetics can mediate T-independent or T-dependent responses. Multivalent peptide mimeotopes can induce high IgM/IgG ratios, as non-conjugated carbohydrates do. Here we observe that immunization with multivalent peptide mimeotope conjugated to BSA enhances carbohydrate reactive antibodies in Balb/c mice and xid mice, with IgG1 greater than IgG2a, in xid mice. These results suggest that mimeotope-conjugate formulations might augment carbohydrate-specific immune responses in immuno-compromised hosts.

Current concepts in cancer vaccine strategies.

Cancer vaccines are entering a new phase of popularity, in part because of the recognition of when a therapeutic vaccine is most effective and the identification of appropriate target antigens. New technologies, most notably gene transfection into dendritic cell and DNA vaccination approaches, have spurred further clinical evaluations. While many researchers consider humoral responses as not being viable for large tumors, these responses may play a role in regulating micrometastases (i.e., adjuvant setting). The recent approval of antibodies as therapeutics for cancer treatment has lent to the viability of this therapy concept. The success of carbohydrate-conjugate vaccines in bacterial systems has also renewed interest in developing such vaccines for cancer immunotherapy. Carbohydrates can be further converted into peptide/protein mimetics with several of these mimetics in clinical trials. These mimetic forms can be manipulated into DNA vaccine types that may be combined into DNA cassettes that contain CTL-associated epitopes to further define a novel strategy for future vaccine development.

Immunological characterization of peptide mimetics of carbohydrate antigens in vaccine design strategies.

Targeting antigens which cannot be readily addressed by genetic vectors is a major challenge in vaccine design. The inter-conversion of carbohydrate antigens into peptide mimetic forms provides a means to broaden the immune response to carbohydrate antigens. Peptides that mimic carbohydrate antigens offer new possibilities to augment immune responses to such antigens that include inducing carbohydrate reactive T-cell responses. Peptide mimeotopes can be formulated in a variety of ways that include multiple antigen peptides (MAP) and as DNA vaccines that prime for different antibody isotypes. On the immunological side we observe that: (i) depending on the immunogen formulation peptide mimetics can be processed by either CD5+ or CD5-B cells; (ii) peptide mimeotope immunization can induce cross-reactive responses to multiple carbohydrate forms; (iii) priming with peptide mimeotopes can enhance carbohydrate immune responses upon boosting and (iv) immunization with peptide mimeotopes can induce carbohydrate reactive T cells.

Exploiting molecular mimicry: defining rules of the game.

Molecular mimicry has been touted as a mean to develop new generation of vaccines to target carbohydrate antigens on pathogens and on tumor cells. Structural and immunological rules governing molecular mimicry require definition for its successful exploitation. Of interest are the kinds of structures that peptides adopt as carbohydrate mimics, the extent to which topological or sequence similarities among peptide mimeotopes define serum cross-reactivity to carbohydrate antigens and the extent to which peptide mimeotopes affect T-cell responses. Rational design concepts can be applied to define how a peptide may mimic carbohydrate antigens, similarities in binding affinities of antibodies for carbohydrate and for peptides, how peptides can mimic core structures on otherwise dissimilar carbohydrate antigens, and how peptide mimeotopes can be used to manipulate cellular responses not achievable by carbohydrate antigens.

Epithelial sodium channel pore region. structure and role in gating.

Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd(2+) was determined. Selected mutants within the amino-terminal portion (alphaVal(569)-alphaTrp(582)) of the pore region responded to MTSEA, MTSET, or Cd(2+) with stimulation or inhibition of whole cell Na(+) current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha-helix. The activation of alphaS580Cbetagamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alphaSer(583)-alphaSer(592)) of the pore region, only one mutation (alphaS583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd(2+), whereas several other mutant channels were partially blocked by MTSEA or Cd(2+) but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.

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.

Selection of an immunogenic and protective epitope of the PsaA protein of Streptococcus pneumoniae using a phage display library.

Streptococcus pneumoniae is an important pathogen that causes disease in young and elderly individuals. The currently available polysaccharide vaccines have limited efficacy in those age groups most susceptible to pneumococcal infections. This study focuses on mapping the epitopes of a surface protein of S. pneumoniae by biopanning a 15 mer phage display library using 5 different monoclonal antibodies (MAbs) against the Pneumoccal surface adhesin A (PsaA). PsaA is a component of the bacterial cell wall that is highly species specific and is involved in bacterial adherence and virulence. Biopanning of the phage display library reveals three distinct epitopes on the PsaA protein. The sequence homology of these epitopes ranges from two to six amino acids when compared to the native PsaA protein type 2. Two of these epitopes have been evaluated for their immunogeneicity in mice. The peptide selected by the MAbs 8G12, 6F6, and 1B7 is referred to as the consensus peptide and is immunogenic in mice. Optimal anti-PsaA response is observed in mice immunized with 50microg of the consensus peptide complexed to proteosomes in 1:1 ratio. The anti-PsaA response is significantly lower than the response to the PsaA native protein. The peptide selected by monoclonal antibody 4E9 in its lipidated form is significantly protective in mice challenged with S. pneumoniae serotype 2 when compared to mice immunized with the native protein. These results show that the selected epitopes of PsaA protein are immunogenic and protective in mice. These epitopes need to be evaluated further as alternatives to currently available vaccines.

A molecular basis for functional peptide mimicry of a carbohydrate antigen.

Peptides may substitute for carbohydrate antigens in carbohydrate-specific immunological reactions. Using the recognition properties of an anti-Lewis Y (LeY) antibody, BR55-2, as a model system, we establish a molecular perspective for peptide mimicry by comparing the three-dimensional basis of BR55-2 binding to LeY with the binding of the same antibody to peptides. The peptides compete with LeY, as demonstrated by enzyme-linked immunosorbent assay and Biacore analysis. The computer program LUDI was used to epitope map the antibody-combining site, correlating peptide reactivity patterns. This approach identified amino acids interacting with the same BR55-2 functional residue groups that recognize the Fucalpha(1-3) moiety of LeY. Molecular modeling indicates that the peptides adopt an extended turn conformation within the BR55-2 combining site, serving to overlap the peptides with the LeY spatial position. Peptide binding is associated with only minor changes in BR55-2, relative to the BR55-2-LeY complex. Anti-peptide serum distinguishes the Fucalpha(1-3) from the Fucalpha(1-4) linkage, therefore differentiating difucosylated neolactoseries antigens. These results further confirm that peptides and carbohydrates can bind to the same antibody-binding site and that peptides can structurally and functionally mimic salient features of carbohydrate epitopes.

Peptide mimicking sialyl-Lewis(a) with anti-inflammatory activity.

Peptides mimicking carbohydrate structure sialyl-Lewis a (SA-Le(a)) have been selected from a diverse dodecapeptide library using monoclonal antibody (MAb) NS19-9. Families of peptides with a consensus sequence consisting of three to nine amino acids and peptides that do not show a conserved core amino acid region were identified. Peptide DLWDWVVGKPAG was selected based on the consensus sequence DXXDXXVG shared with other peptides and strong binding in Western blot. Peptide competes with antibody binding to its native carbohydrate antigen, SA-Le(a), at 50% inhibitory concentration (IC(50)), 700 microM, implying that it represents a structural mimic of the carbohydrate epitope recognized by MAb. Statistically significant reduction of neutrophil recruitment into the intraperitoneal cavity was observed upon administration of this peptide in a murine acute inflammation model in vivo. Results suggest that the peptide mimic of SA-Le(a) carbohydrate might bind to E-selectin and block its interaction with another ligand, sialyl-Lewis X (SA-LeX), expressed on neutrophils.

Selection of an immunogenic peptide mimic of the capsular polysaccharide of Neisseria meningitidis serogroup A using a peptide display library.

The presently available meningococcal vaccine is poorly immunogenic in infants and fails to induce long-lasting immunity in adults. Efforts to convert this TI-2 type vaccine into a T dependent vaccine are being actively pursued and include conjugate vaccine development. Alternatively, the meningococcal polysaccharide can be rendered into a T dependent antigen through the use of peptides which mimic the capsular polysaccharide complexed or conjugated to potent protein carrier molecules. We have previously developed an anti-idiotypic monoclonal antibody (mAb) based peptide mimic of meningococcal group C polysaccharide (MCPS). A direct approach to identification of peptide mimics of antigen is through the use of peptide display libraries. We have utilized a phage library and a mAb with specificity for meningococcal group A polysaccharide (MAPS) to screen for a peptide mimic of MAPS. Six different peptide motifs were selected with the use of the mAb. Thirty-eight of the 60 sequenced phage clones were represented by motif 1 and 2 which differed only in three amino acids at the carboxy terminus. Immunological assays were performed. Phage clones with motif 1 and 2 were capable of binding human hyperimmune sera and inhibiting the binding of human hyperimmune sera to nominal antigen. Immunization with motif 1 peptide complexed to proteosomes resulted in an anti-MAPS antibody response. Priming with the peptide proteosome complex induced an anamnestic response indicating the formation of immunological memory.

Mechanism of action of amiloride: a molecular prospective.

Amiloride is a prototypic inhibitor of epithelial sodium channels. Rapid progress has been made in our understanding of the structure of the sodium channel and related cation-selective channels. This work, coupled with experiments examining how selected sodium channel mutations affect amiloride binding, provides critical clues towards defining sites within the channel that bind amiloride. Residues within the channel pore and within its extracellular domain participate in amiloride binding. These results suggest that sites that interact with amiloride within the channel's extracellular domain may be in close proximity to residues within the channel's pore.

Vaccination with carbohydrate peptide mimotopes promotes anti-tumor responses.

Tumor-associated carbohydrate (TAC) antigens are important targets in cancer vaccine efforts. Carbohydrates are, however, frequently poor immunogens, in that they are T-cell-independent antigens. Molecular mimicry of TAC by peptides is an alternative approach to generating anti-carbohydrate immune responses. Here we demonstrate that peptide mimotopes can elicit antibody responses that cross-react with representative human TAC antigens. Primary immunization with such a multiple antigenic peptide, along with QS-21 as adjuvant, elicits cytotoxic antibodies reactive with naturally occurring forms of TAC expressed on tumor cells, and vaccination of mice with peptide mimotopes reduced tumor growth and prolonged host survival in a murine tumor model.

Human immune response to a peptide mimic of Neisseria meningitidis serogroup C in hu-PBMC-SCID mice.

An anti-idiotype-based peptide mimic vaccine for Neisseria meningitidis serogroup C polysaccharide (MCPS) has been developed and shown to induce a response in mice that is specific, functional, and T-dependent. In this study, the immunogenicity of the MCPS peptide mimic vaccine preparation, as a potential vaccine for use in humans, is shown using the hu-PBMC-SCID mouse model. The human antibody response to the MCPS peptide mimic vaccine is specific and functional as shown by inhibition enzyme-linked immunoadsorbent assay (ELISA) and bactericidal assay. These data support the usefulness of the peptide mimic vaccine strategy for humans.

Towards the development of peptide mimotopes of carbohydrate antigens as cancer vaccines.

Tumor-associated carbohydrate antigens are considered important targets in efforts to develop cancer vaccines. To further enhance vaccine efforts, we are developing peptide mimotopes of tumor-associated carbohydrate antigens that can elicit functional immune responses. Mapping peptide epitopes with anticarbohydrate antibodies can lend to defining structural relationships that can go undetected by screening of carbohydrate antigens alone. Here we contrast reactivity patterns for peptides using monoclonal antibodies (MAbs) directed to the neolactoseries related Lewis Y (LeY) and sialyl-Lewis X (sLeX) antigen and the GD3/GD2 ganglioside antigen. We observe that representative MAbs cross-react with a WRY-containing peptide and that this motif type is isolated by the respective monoclonal in peptide phage display screening. Primary immunization with multiple antigen peptide preparations with QS-21 adjuvant efficiently elicited cytotoxic IgM antibodies for a murine Meth A fibrosarcoma line expressing sLeX. The cytotoxicity of IgG polyclonal response was found to be as effective as IgM in mediating complement-dependent cytotoxicity against the Meth A line. These experiments suggest that peptide mimotopes of the LeY and sLeX tumor-associated carbohydrate antigen and QS-21 adjuvant could be considered as an immunogenic therapeutic vaccine in carcinoma and melanoma patients in the minimal residual disease setting.

Antiidiotypic antibody recognizes an amiloride binding domain within the alpha subunit of the epithelial Na+ channel.

We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na+ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na+ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis alphaENaC. RA6.3 specifically recognized an amiloride binding domain within the alpha-subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the alpha-subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to alphaENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within alphaENaC.