A nontoxic mutant of cholera toxin elicits Th2-type responses for enhanced mucosal immunity

We have characterized a nontoxic mutant of cholera toxin (CT) as a mucosal adjuvant in mice. The mutant CT was made by substitution of serine with phenylalanine at position 61 of the A subunit (S61F), which resulted in loss of ADP ribosyltransferase activity and toxicity. Mice were intranasally immunized with ovalbumin, tetanus toxoid, or influenza virus either alone or together with mutant CT S61F, native CT, or recombinant CT-B. Mice immunized with these proteins plus S61F showed high serum titers of protein-specific IgG and IgA antibodies that were comparable to those induced by native CT. Further, high protein-specific IgA antibody responses were observed in nasal and vaginal washes, saliva, and fecal extracts as well as increased numbers of IgG and IgA antibody forming cells in cervical lymph nodes and lung tissues of mice intranasally immunized with these proteins and S61F or native CT, but not with recombinant CT-B or protein alone. Both S61F and native CT enhanced the induction of ovalbumin-specific CD4+ T cells in lung and splenic tissues, and these T cells produced a Th2-type cytokine pattern of interleukin 4 (IL-4), IL-5, IL-6, and IL-10 as determined by analysis of secreted proteins and by quantitation of cytokine-specific mRNA. These results have shown that mutant CT S61F is an effective mucosal adjuvant when administrated intranasally and induces mucosal and systemic antibody responses which are mediated by CD4+ Th2-type cells.

Limited internal friction in the rate-limiting step of a two-state protein folding reaction

Small, single-domain proteins typically fold via a compact transition-state ensemble in a process well fitted by a simple, two-state model. To characterize the rate-limiting conformational changes that underlie two-state folding, we have investigated experimentally the effects of changing solvent viscosity on the refolding of the IgG binding domain of protein L. In conjunction with numerical simulations, our results indicate that the rate-limiting conformational changes of the folding of this domain are strongly coupled to solvent viscosity and lack any significant “internal friction” arising from intrachain collisions. When compared with the previously determined solvent viscosity dependencies of other, more restricted conformational changes, our results suggest that the rate-limiting folding transition involves conformational fluctuations that displace considerable amounts of solvent. Reconciling evidence that the folding transition state ensemble is comprised of highly collapsed species with these and similar, previously reported results should provide a significant constraint for theoretical models of the folding process.

Expression cloning of cDNA encoding a human β-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis

The structure and biosynthesis of poly-N-acetyllactosamine display a dramatic change during development and oncogenesis. Poly-N-acetyllactosamines are also modified by various carbohydrate residues, forming functional oligosaccharides such as sialyl Lex. Herein we describe the isolation and functional expression of a cDNA encoding β-1,3-N-acetylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-N-acetyllactosamine. For this expression cloning, Burkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA libraries derived from human melanoma and colon carcinoma cells. Transfected Namalwa cells overexpressing the i antigen were continuously selected by fluorescence-activated cell sorting because introduced plasmids containing Epstein–Barr virus replication origin can be continuously amplified as episomes. Sibling selection of plasmids recovered after the third consecutive sorting resulted in a cDNA clone that directs the increased expression of i antigen on the cell surface. The deduced amino acid sequence indicates that this protein has a type II membrane protein topology found in almost all mammalian glycosyltransferases cloned to date. iGnT, however, differs in having the longest transmembrane domain among glycosyltransferases cloned so far. The iGnT transcript is highly expressed in fetal brain and kidney and adult brain but expressed ubiquitously in various adult tissues. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the enzyme responsible for the formation of GlcNAcβ1 → 3Galβ1 → 4GlcNAc → R structure and poly-N-acetyllactosamine extension.

T cell functions in granulocyte/macrophage colony-stimulating factor deficient mice

Immunological functions were analyzed in mice lacking granulocyte/macrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to allo-antigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF +/+ and GM-CSF −/− mice. To investigate the responses of CD8+ and CD4+ T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD8+ T cells with specificity for ovalbumin peptide could not be induced in GM-CSF −/− mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF −/− mice. Purified CD4+ T cells from GM-CSF −/− mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-γ (IFN-γ) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD4+ T cells from GM-CSF −/− mice proliferated as well as those from GM-CSF +/+ mice and produced high amounts of IFN-γ and IL-4. To analyze the rescue effect of DC on CD4+ T cells, supernatants from (i) CD4+ T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD4+ T cells and KLH-pulsed spleen cells from GM-CSF −/− mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-γ production of CD4+ T cells from GM-CSF −/− mice.

DNA immunization: Induction of higher avidity antibody and effect of route on T cell cytotoxicity

Immunizations of mice with plasmid DNAs encoding ovalbumin (OVA), human Ig, and hen egg lysozyme were compared with doses of soluble protein (without adjuvant) that induced similar IgG responses. The route of immunization influenced the magnitude of the antibody (Ab) response in that intradermal (i.d.) injection elicited higher IgG Ab levels than i.m. injection in both DNA- and protein-immunized mice. Although total IgG levels were similar to soluble protein controls, the avidity of the anti-OVA Abs generated by DNA immunization were 100- and 1,000-fold higher via the i.m. or i.d. route, respectively. However, despite the generation of high-avidity Ab in DNA-immunized mice, germinal centers could not be detected in either DNA- or protein-immunized mice. Examination of the IgG subclass response showed that IgG2a was induced by i.m. DNA immunization, coinciding with elevated interferon γ production, whereas a dominant and elevated IgG1 response, coinciding with detectable interleukin 4 production, was generated after i.d. immunization with DNA or soluble OVA and hen egg lysozyme but not human Ig protein. As expected, cytotoxic T cell (CTL) responses could be detected only after DNA immunization. I.d. immunization produced the strongest CTL responses early (2 weeks) but was similar to i.m. later. Therefore, DNA immunization can differ from protein immunization by its ability to induce rapid CTL responses and higher avidity Ab, both of which are advantageous for vaccination.

Ocular cicatricial pemphigoid antigen: Partial sequence and biochemical characterization

Ocular cicatricial pemphigoid (OCP) is an autoimmune disease that affects mainly conjunctiva and other squamous epithelia. OCP is histologically characterized by a separation of the epithelium from underlying tissues within the basement membrane zone. Immunopathological studies demonstrate the deposition of anti-basement membrane zone autoantibodies in vivo. Purified IgG from sera of patients with active OCP identified a cDNA clone from a human keratinocyte cDNA library that had complete homology with the cytoplasmic domain of β4-integrin. The sera recognized a 205-kDa protein in human epidermal, human conjunctiva, and tumor cell lysates that was identified as β4-integrin by its reaction with polyclonal and monoclonal antibodies to human β4-integrin. Sera from patients with bullous pemphigoid, pemphigus vulgaris, and cicatricial pemphigoid-like diseases did not recognize the 205-kDa protein, indicating the specificity of the binding. These data strongly implicate a role for human β4-integrin in the pathogenesis of OCP. It should be emphasized that multiple antigens in the basement membrane zone of squamous epithelia may serve as targets for a wide spectrum of autoantibodies observed in vesiculobullous diseases. Molecular definition of these autoantigens will facilitate the classification and characterization of subsets of cicatricial pemphigoid and help distinguishing them from bullous pemphigoid. This study highlights the function and importance of β4-integrin in maintaining the attachment of epithelial cells to the basement membrane.

Antigenicity of fullerenes: Antibodies specific for fullerenes and their characteristics

The recent interest in using Buckminsterfullerene (fullerene) derivatives in biological systems raises the possibility of their assay by immunological procedures. This, in turn, leads to the question of the ability of these unprecedented polygonal structures, made up solely of carbon atoms, to induce the production of specific antibodies. Immunization of mice with a C60 fullerene derivative conjugated to bovine thyroglobulin yielded a population of fullerene-specific antibodies of the IgG isotype, showing that the immune repertoire was diverse enough to recognize and process fullerenes as protein conjugates. The population of antibodies included a subpopulation that crossreacted with a C70 fullerene as determined by immune precipitation and ELISA procedures. These assays were made possible by the synthesis of water-soluble fullerene derivatives, including bovine and rabbit serum albumin conjugates and derivatives of trilysine and pentalysine, all of which were characterized as to the extent of substitution and their UV-Vis spectra. Possible interactions of fullerenes with the combining sites of IgG are discussed based on the physical chemistry of fullerenes and previously described protein-fullerene interactions. They remain to be confirmed by the isolation of mAbs for x-ray crystallographic studies.

Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells

The class B, type I scavenger receptor, SR-BI, binds high density lipoprotein (HDL) and mediates the selective uptake of HDL cholesteryl ester (CE) by cultured transfected cells. The high levels of SR-BI expression in steroidogenic cells in vivo and its regulation by tropic hormones provides support for the hypothesis that SR-BI is a physiologically relevant HDL receptor that supplies substrate cholesterol for steroid hormone synthesis. This hypothesis was tested by determining the ability of antibody directed against murine (m) SR-BI to inhibit the selective uptake of HDL CE in Y1-BS1 adrenocortical cells. Anti-mSR-BI IgG inhibited HDL CE-selective uptake by 70% and cell association of HDL particles by 50% in a dose-dependent manner. The secretion of [3H]steroids derived from HDL containing [3H]CE was inhibited by 78% by anti-mSR-BI IgG. These results establish mSR-BI as the major route for the selective uptake of HDL CE and the delivery of HDL cholesterol to the steroidogenic pathway in cultured mouse adrenal cells.

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Toward optimized carbohydrate-based anticancer vaccines: Epitope clustering, carrier structure, and adjuvant all influence antibody responses to Lewisy conjugates in mice

The feasibility of using carbohydrate-based vaccines for the immunotherapy of cancer is being actively explored at the present time. Although a number of clinical trials have already been conducted with glycoconjugate vaccines, the optimal design and composition of the vaccines has yet to be determined. Among the candidate antigens being examined is Lewisy (Ley), a blood group-related antigen that is overexpressed on the majority of human carcinomas. Using Ley as a model for specificity, we have examined the role of epitope clustering, carrier structure, and adjuvant on the immunogenicity of Ley conjugates in mice. A glycolipopeptide containing a cluster of three contiguous Ley-serine epitopes and the Pam3Cys immunostimulating moiety was found to be superior to a similar construct containing only one Ley-serine epitope in eliciting antitumor cell antibodies. Because only IgM antibodies were produced by this vaccine, the effect on immunogenicity of coupling the glycopeptide to keyhole limpet hemocyanin was examined; although both IgM and IgG antibodies were formed, the antibodies reacted only with the immunizing structure. Reexamination of the clustered Ley-serine Pam3Cys conjugate with the adjuvant QS-21 resulted in the identification of both IgG and IgM antibodies reacting with tumor cells, thus demonstrating the feasibility of an entirely synthetic carbohydrate-based anticancer vaccine in an animal model.

Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate: A phase I trial

The carbohydrate antigen globo H commonly found on breast cancer cells is a potential target for vaccine therapy. The objectives of this trial were to determine the toxicity and immunogenicity of three synthetic globo H-keyhole limpet hemocyanin conjugates plus the immunologic adjuvant QS-21. Twenty-seven metastatic breast cancer patients received five vaccinations each. The vaccine was well tolerated, and no definite differences were observed among the three formulations. Serologic analyses demonstrated the generation of IgM antibody titers in most patients, with minimal IgG antibody stimulation. There was significant binding of IgM antibodies to MCF-7 tumor cells in 16 patients, whereas IgG antibody reactivity was observed in a few patients. There was evidence of complement-dependent cytotoxicity in several patients. Affinity column purification supported the specificity of IgM antibodies for globo H. On the basis of these data, globo H will constitute one component of a polyvalent vaccine for evaluation in high-risk breast cancer patients.

Peyer's patches are required for oral tolerance to proteins

To clarify the role of Peyer's patches in oral tolerance induction, BALB/c mice were treated in utero with lymphotoxin β-receptor Ig fusion protein to generate mice lacking Peyer's patches. When these Peyer's patch-null mice were fed 25 mg of ovalbumin (OVA) before systemic immunization, OVA-specific IgG Ab responses in serum and spleen were seen, in marked contrast to low responses in OVA-fed normal mice. Further, high T-cell-proliferative- and delayed-type hypersensitivity responses were seen in Peyer's patch-null mice given oral OVA before systemic challenge. Higher levels of CD4+ T-cell-derived IFN-γ, IL-4, IL-5, and IL-10 syntheses were noted in Peyer's patch-null mice fed OVA, whereas OVA-fed normal mice had suppressed cytokine levels. In contrast, oral administration of trinitrobenzene sulfonic acid (TNBS) to Peyer's patch-null mice resulted in reduced TNBS-specific serum Abs and splenic B cell antitrinitrophenyl Ab-forming cell responses after skin painting with picryl chloride. Further, when delayed-type hypersensitivity and splenic T cell proliferative responses were examined, Peyer's patch-null mice fed TNBS were unresponsive to hapten. Peyer's patch-null mice fed trinitrophenyl-OVA failed to induce systemic unresponsiveness to hapten or protein. These findings show that organized Peyer's patches are required for oral tolerance to proteins, whereas haptens elicit systemic unresponsiveness via the intestinal epithelial cell barrier.

Quantitative Intercellular Localization of NADH-Dependent Glutamate Synthase Protein in Different Types of Root Cells in Rice Plants1

The quantitative analysis with immunogold-electron microscopy using a single-affinity-purified anti-NADH-glutamate synthase (GOGAT) immunoglobulin G (IgG) as the primary antibody showed that the NADH-GOGAT protein was present in various forms of plastids in the cells of the epidermis and exodermis, in the cortex parenchyma, and in the vascular parenchyma of root tips (<10 mm) of rice (Oryza sativa) seedlings supplied with 1 mm NH4+ for 24 h. The values of the mean immunolabeling density of plastids were almost equal among these different cell types in the roots. However, the number of plastids per individual cell type was not identical, and some parts of the cells in the epidermis and exodermis contained large numbers of plastids that were heavily immunolabeled. Although there was an indication of labeling in the mitochondria using the single-affinity-purified anti-NADH-GOGAT IgG, this was not confirmed when a twice-affinity-purified IgG was used, indicating an exclusively plastidial location of the NADH-GOGAT protein in rice roots. These results, together with previous work from our laboratory (K. Ishiyama, T. Hayakawa, and T. Yamaya [1998] Planta 204: 288–294), suggest that the assimilation of exogeneously supplied NH4+ ions is primarily via the cytosolic glutamine synthetase/plastidial NADH-GOGAT cycle in specific regions of the epidermis and exodermis in rice roots. We also discuss the role of the NADH-GOGAT protein in vascular parenchyma cells.

CML66, a broadly immunogenic tumor antigen, elicits a humoral immune response associated with remission of chronic myelogenous leukemia

This report describes a tumor-associated antigen, termed CML66, initially cloned from a chronic myelogenous leukemia (CML) cDNA expression library. CML66 encodes a 583-aa protein with a molecular mass of 66 kDa and no significant homology to other known genes. CML66 gene is localized to human chromosome 8q23, but the function of this gene is unknown. CML66 is expressed in leukemias and a variety of solid tumor cell lines. When examined by Northern blot, expression in normal tissues was restricted to testis and heart, and no expression was found in hematopoietic tissues. When examined by quantitative reverse transcription–PCR, expression in CML cells was 1.5-fold higher than in normal peripheral blood mononuclear cells. The presence of CML66-specific antibody in patient serum was confirmed by Western blot and the development of high titer IgG antibody specific for CML66 correlated with immune induced remission of CML in a patient who received infusion of normal donor lymphocytes for treatment of relapse. CML66 antibody also was found in sera from 18–38% of patients with lung cancer, melanoma, and prostate cancer. These findings suggest that CML66 may be immunogenic in a wide variety of malignancies and may be a target for antigen-specific immunotherapy.

Patterning of functional antibodies and other proteins by photolithography of silane monolayers.

We have demonstrated the assembly of two-dimensional patterns of functional antibodies on a surface. In particular, we have selectively adsorbed micrometer-scale regions of biotinylated immunoglobulin that exhibit specific antigen binding after adsorption. The advantage of this technique is its potential adaptability to adsorbing arbitrary proteins in tightly packed monolayers while retaining functionality. The procedure begins with the formation of a self-assembled monolayer of n-octadecyltrimethoxysilane (OTMS) on a silicon dioxide surface. This monolayer can then be selectively removed by UV photolithography. Under appropriate solution conditions, the OTMS regions will adsorb a monolayer of bovine serum albumin (BSA), while the silicon dioxide regions where the OTMS has been removed by UV light will adsorb less than 2% of a monolayer, thus creating high contrast patterned adsorption of BSA. The attachment of the molecule biotin to the BSA allows the pattern to be replicated in a layer of streptavidin, which bonds to the biotinylated BSA and in turn will bond an additional layer of an arbitrary biotinylated protein. In our test case, functionality of the biotinylated goat antibodies raised against mouse immunoglobulin was demonstrated by the specific binding of fluorescently labeled mouse IgG.