Different Glycosylation Pattern of Human IgG1 and IgG3 Antibodies Isolated from Transiently as well as Permanently Transfected Cell Lines

The effector functions of IgG depend on the presence of carbohydrates attached to asparagine 297 in the Fc‐portion. In this report, glycosylation profiles of recombinant wild‐type and mutant IgG1 and IgG3 antibodies produced from three cell lines were analysed using LC‐ESI‐Orbitrap. Clear differences were detected between IgG1 and IgG3 glycoforms, where IgG1 generally contained fucosylated glycoforms, whilst IgG3 mainly were non‐fucosylated. When using NS‐0 and J558L cells for permanent transfection, IgG1 wt glycoforms differed between the two cell lines, whilst IgG3 wt glycoforms did not. Transiently transfected HEK 293E cells were used to produce IgG1 and IgG3 wt and mutants, affecting complement activation. Cell supernatants were harvested at early and late time points and analysed separately. IgGs harvested late showed simpler and less developed glycosylation structure compared to those harvested early. The IgG harvested early was slightly more effective in complement activation than those harvested late, whilst the antibody‐dependent cell‐mediated cytotoxicity was unaltered. Generally, the glycosylation pattern of the mutants tested, including a hinge truncate mutant of IgG3, did not differ significantly from the wild‐type IgGs. The striking difference in glycosylation pattern of IgG1 compared to IgG3 therefore appears not to be due to the long hinge region of IgG3 (62 amino acids) relative to the IgG1 hinge region (15 amino acids). Furthermore, mutation variants at or near the C1q binding site showed similar glycosylation structure and difference in their complement activation activity observed earlier is thus most likely due to differences in protein structure only.     

Different Glycosylation Pattern of Human IgG1 and IgG3 Antibodies Isolated From Transiently as Well as Permanently Transfected Cell Lines

The effector functions of IgG depend on the presence of carbohydrates attached to asparagine 297 in the Fc portion. In this report, glycosylation profiles of recombinant wild‐type and mutant IgG1 and IgG3 antibodies produced from three cell lines were analysed using LC‐ESI‐Orbitrap. Differences were detected between IgG1 and IgG3, where IgG1 generally contained fucosylated glycoforms, while IgG3 showed a noticeable percentage of non‐fucosylated glycoforms. When using NS‐0 and J558L cells for permanent transfection, IgG1 wt glycoforms differed between the two cell lines, while IgG3 wt glycoforms did not. Transiently transfected HEK 293E cells were used to produce IgG1 and IgG3 wt and mutants, affecting complement activation. Cell supernatants were harvested at early and late time points and analysed separately. IgG's harvested late showed simpler and less developed glycosylation structure compared with those harvested early. IgG's harvested early were slightly more effective in complement activation than those harvested late, while the antibody‐dependent cell‐mediated cytotoxicity was unaltered. Generally, the glycosylation pattern of the mutants tested, including a hinge truncate mutant of IgG3, did not differ significantly from the wild‐type IgG's. The difference in glycosylation pattern of IgG1 compared with IgG3 therefore appears not to be due to the long hinge region of IgG3 (62 amino acids) relative to the IgG1 hinge region (15 amino acids). Furthermore, mutation variants at or near the C1q‐binding site showed similar glycosylation structure and difference in their complement activation activity observed earlier is thus most likely due to differences in protein structure only.     

RASA, a recombinant single-chain variable fragment (scFv) antibody directed against the human sperm surface: implications for novel contraceptives

BACKGROUND: A recombinant single-chain variable fragment (scFv) antibody was engineered to a tissue-specific carbohydrate epitope located on human sperm agglutination antigen-1 (SAGA-1), a sperm glycoform of CD52. METHODS AND RESULTS: cDNAs encoding the variable regions of the S19 [IgG(1)kappa] monoclonal antibody (mAb) were identified, linked, and cloned into the pCANTAB 5E vector. The recombinant anti-sperm antibody (RASA) was expressed in E. coli HB2151 cells as a 29 kDa monomer and, remarkably, also formed multimers of approximately 60 and 90 kDa. RASA reacted with the endogenous SAGA-1 antigen by Western blot analysis, labelled the entire human sperm surface by indirect immunofluorescence, and aggregated human spermatozoa in a tangled (head-to-head, head-to-tail, tail-to-tail) pattern of agglutination, as was also observed with the native S19 mAb. CONCLUSIONS: These results demonstrate that active recombinant antibodies can be produced to a tissue-specific carbohydrate epitope on the human sperm surface, thereby opening opportunities for novel contraceptive agents.     

A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies

Monoclonal antibodies (Mab) are the fastest growing group of biopharmaceuticals in development. For production in mammalian cells, the four polypeptide chains of the immunoglobulin diheterotetramer must be assembled prior to exit from the endoplasmic reticulum. Various recombinant Mab expression vectors have been developed utilizing mono-and bicistronic expression cassettes encoded on one or two plasmids. However, there are only few studies providing information on the type of vector design optimal for stable or transient production of recombinant IgG. Consequently, in this study, we have constructed a series of mammalian expression vectors for the production of recombinant human or chimeric IgG antibodies with different expression cassette designs. Versions for monocistronic and bicistronic expression with different promoters and cistron arrangements were generated. Antibody production levels were evaluated in transiently transfected 293T and CHO-K1 cells. Furthermore, stable CHO cell lines were generated and analyzed for antibody production levels and stability. Our results indicate that compared to monocistronic expression, EMCV IRES-mediated bicistronic expression constructs yield similar antibody expression levels and show long-term stability in CHO cell lines. Addition of a third cistron encoding YFP was shown to facilitate screening and isolation of clones using a FACS sorter.     

Blood clearance in the rat of a recombinant mouse monoclonal antibody lacking the N-linked oligosaccharide side chains of the CH2 domains.

The serum half-lives of a wild-type recombinant mouse monoclonal antibody of the IgG2b isotype and a mutant antibody differing from the wild-type antibody by a single amino acid substitution introduced into the CH2 domain, the replacement of Asn 297 by Ala to delete the conserved site of heavy chain glycosylation, were determined in the rat. The biological half-life of the aglycosyl Asn 297-Ala mutant recombinant antibody (4.8 days) was significantly shorter than that of the normally glycosylated wild-type antibody (7.4 days) by enzyme immunoassay. A similar difference between the biological half-lives of 125I-labelled aglycosyl and wild-type antibodies (2.9 and 4.0 days, respectively) was determined by gamma counting. Analysis of serum samples demonstrated that both recombinant antibodies were present in the circulation predominantly as intact monomeric IgG and revealed no differences that could account for the more rapid elimination of the aglycosyl antibody. The results of this investigation indicate that the carbohydrate residues contribute only in part to the survival of IgG in vivo and suggest that the diminished half-life of the aglycosyl antibody is due to increased catabolism in the extravascular tissues.     

Evaluation of the adjuvant effect of Escherichia coli heat-labile enterotoxin mutant (LTK63) on the systemic immune responses to intranasally co-administered measles virus nucleoprotein. Part I: antibody responses

The adjuvanticity and immunogenicity of the heat-labile enterotoxin (LT) of Escherichia coli and of its non-toxic mutant, LTK63, was evaluated after intranasal administration of CBA mice with recombinant measles virus nucleoprotein (rMVNP) with or without LT or LTK63. Both LT and LTK63 were shown to be highly immunogenic with higher responses observed 4 weeks after the booster immunization. Although the nucleoprotein was immunogenic on its own, mice immunized with the nucleoprotein plus wild type LT produced significantly high antibody responses (p<0.01). Mice that received the rMVNP with LTK63 also generated strong antibody responses to rMVNP. These antibodies were also significantly higher than those of rMVNP alone (p< 0.05). No significant differences were observed between groups of mice immunized intranasally with rMVNP plus LT or LTK63 (p> 0.05). Data on IgG antibody isotype profiles showed that IgG 1 and IgG 2a were predominant in mice immunized with rMVNP + LT or LTK63 whereas IgG 1 predominated when rMVNP was given on its own implying that LT and LTK63 induce both Th1 and Th2-type immune responses. These results highlight the great potential of this non-toxic mutant of LT as a safe vaccine adjuvant.     

Neisseria meningitidis Native Outer Membrane Vesicles Containing Different Lipopolysaccharide Glycoforms as Adjuvants for Meningococcal and Nonmeningococcal Antigens

We evaluated the adjuvant effect of a modified glycoform of lipopolysaccharide (LPS) (LgtB-LpxL1) compared to that of the nonmodified glycoform Lpxl1 serogroup B meningococcal H44/76 native outer membrane vesicles (nOMVs) on immune responses to vaccination with the recombinant meningococcal protein, rPorA, tetanus toxoid, or meningococcal serogroup C capsular polysaccharide. We used LgtB-LpxL1 LPS because the disruption of the lgtB gene, which results in the exposure of N-acetylglucosamine-galactose-glucose residues in the LPS outer core, has been shown to enhance the activation of human dendritic cells in vitro. The responses were compared to those of a monophosphoryl lipid A (MPL)-based adjuvant and to an aluminum hydroxide suspension. The nOMVs induced blood serum IgG responses against each of the three antigens comparable to those obtained with MPL or aluminum salt. However, nOMVs elicited (i) a lower IgG1/IgG2a ratio against rPorA and (ii) serum bactericidal antibody titers superior to those achieved with aluminum salt, reaching similar titers to those obtained with MPL. Similarly, bactericidal antibody titers induced by immunization with meningococcal serogroup C polysaccharide and nOMVs were similar to those obtained using MPL but were better than those with aluminum salt. Immunization with tetanus toxoid and nOMVs resulted in tetanus toxoid-specific IgG responses similar to those obtained when adjuvanted with aluminum salt. These results highlight the potential utility of meningococcal LpxL1 LPS-containing nOMVs as an adjuvant for recombinant meningococcal protein vaccines and suggest their possible use with a variety of other antigens.     

Antibody-dependent cytotoxicity mediated by natural killer cells is enhanced by castanospermine-induced alterations of IgG glycosylation

Inhibitors of glycosylation and carbohydrate processing were used to probe the functional consequences of specific, differential alterations in glycosylation of monoclonal IgG secreted by hybridoma clones. Neither the absence of glycosylation nor the presence of atypical oligosaccharides significantly influenced binding of the monoclonal antibody to the cell surface antigen recognized. However, lymphocyte-mediated antibody-dependent cytotoxicity was enhanced significantly, as compared to native (unmodified) IgG-sensitized target cells, when target cells were sensitized with IgG bearing the a typical oligosaccharides induced metabolically by castanospermine. N-methyldeoxynojirimycin, deoxymannojirimycin or monesin, but not by swainsonine. The enhanced cytotoxicity was mediated by natural killer cells but not by monocytes or interferon-activated polymorphonuclear leukocytes. By contrast, antibodydependent cytotoxicity mediated by activated polymorphonuclear leukocytes against target cells sensitized with the IgG glycosylation phenotypes induced by swainsonine and tunicamycin. but not by castanospermine, was decreased in comparison to cytotoxicity against target cells sensitized with native IgG.

The enhanced lymphocyte-mediated cytotoxicity was Fc receptor-dependent.

A panel of monoclonal antibodies directed against different human tumor target cells was used to demonstrate that the castanospermine-induced IgG phenotype generally enhanced antibody-dependent tumoricidal activity mediated by natural killer cells. However, differences in lymphocyte response to an alteration in IgG glycosylation were observed.     

Possible role of IL-6 in pathogenesis of immune complex-mediated glomerulonephritis in NZB/W F1 mice: induction of IgG class anti-DNA autoantibody production

This study demonstrates that interleukin-6 (IL-6) increases the autoantibody production by B cells from NZB/W F1 mice. Splenic B cells were cultured for 5 days in the presence or absence of human IL-6, and then the anti-DNA antibody and immunoglobulin contents in the culture supernatants were measured by ELISA. Adding IL-6 increased IgG anti-DNA antibody production by B cells from old mice (30 weeks), but not from young ones (17 weeks). B cells obtained from both young and old mice produced IgM anti-DNA antibody, which increased when IL-6 was added. The increased anti-DNA antibody production was suppressed by anti-recombinant human IL-6 antibody to the background level, i.e. antibody contents in the absence of IL-6. In contrast, murine IL-5 did not increase IgG anti-DNA antibody production, although it promoted the production of IgM anti-DNA antibody. Furthermore, when IL-5 was added in combination with IL-6, there was an additive increase in IgM, but not in IgG anti-DNA antibody production. Similar results were obtained in the measurement of the immunoglobulin contents. These results suggest the possible role of IL-6 in the pathogenesis of autoimmune disease in NZB/W F1 mice.     

Further evidence for preferential production of IgG2 anti-DNP antibody in guinea pigs immunized with DNP-Escherichia coli

As reported previously, radioimmunoelectrophoretic analysis of guinea pig antisera to 2,4-dinitrophenylated cells of Escherichia coli (DNP-E. coli) revealed that synthesis of antibodies to 2,4-dinitrophenyl residues (DNP) was restricted to one of the IgG antibodies, IgG2, when guinea pigs were immunized with the antigen in both the presence and absence of Freund's adjuvants. In order to confirm further this unique antibody response to DNP-E. coli, the subclass of anti-DNP antibodies produced was analyzed by DEAE-cellulose chromatography, electrofocusing and determination of antibody activities. The results so far obtained indicated that about 97% of the antibodies produced was of the IgG2 subclass and only a trace of the IgG1 antibody was produced, independently of the mode of immunization, though a small amount of IgG1 anti-DNP antibody was produced concomitantly on increasing the immunizing dose to 3.0 mg in saline. Furthermore, the cell-free extract prepared from DNP-E. coli also induced the preferential production of IgG2 anti-DNP antibody.     

In vitro induction of IgG anti-DNA antibody from high density B cells of systemic lupus erythematosus patients by an HLA DR-restricted T cell clone.

An HLA-DR restricted T cell clone (26G11) which recognized a lymphoid cell-derived autoantigen associated with DR4 molecule was shown to induce not only autologous but also allogenic DR4+ B cells to produce large amounts of antibodies of the IgG and IgM classes. Using the helper activity of this clone, we investigated the mechanism of anti-DNA antibody production in DR-matched patients with systemic lupus erythematosus (SLE). When cultured with 26G11 cells, B cells from DR-matched normal control subjects produced large amounts of IgM anti-DNA antibody, but did not produce IgG anti-DNA antibody which is thought to have a pathological role in SLE. In contrast, B cells from DR-matched patients with active SLE spontaneously produced a fairly large amount of IgG anti-DNA antibody, and the production was augmented by the T cell clone. Little IgG anti-DNA antibody was produced by the B cells of patients with inactive SLE in either the presence or absence of T cell clone. We next fractionated B cells into low density B (LD-B) and high density B (HD-B) cells by centrifugation on discontinuous Percoll density gradients. IgG anti-DNA antibody was spontaneously produced by LD-B cells of active SLE patients but not by those either of inactive SLE patients or normal controls. On the other hand, although IgG anti-DNA antibody was not spontaneously produced by the HD-B cells of both active and inactive SLE patients, it could easily be induced by their culture with the T cell clone. Our results clearly show the existence of IgG anti-DNA antibody-producing B cells in the peripheral blood of SLE patients irrespective of their disease activity and suggest that autoreactive T cells may play a pathogenic role in SLE through the induction of autoantibody production.     

Expression of neutralizing recombinant human antibodies against Varicella Zoster virus for use as a potential prophylactic

Chickenpox is a highly infectious disease that can be life-threatening to certain groups such as the newborn of nonimmune mothers and immunocompromised patients. At present, prophylactic treatment of individuals at risk involves the use of a polyclonal antibody preparation derived from the pooled sera of hyperimmune donors. While this product is effective, there are problems associated with maintaining supply, which depends on the availability of donors, and the variation of potency between batches. An effective human monoclonal preparation would be of value by providing a well-characterized and standardized preparation available on demand. In this study recombinant human anti-varicella zoster virus (VZV) monoclonals were generated from the mRNA of unstable anti-VZV secreting heterohybridoma cell lines, and characterized according to their molecular weight, isoelectric point, glycosylation, binding to C1q, and efficacy at neutralizing VZV in vitro. In one antibody (AEVZ 5.3) the VH region was grafted from the IgG1 parent antibody onto an IgG3 backbone to determine the effect of isotype on neutralization in vitro. Antibodies were expressed from NSO cells at concentrations of 3-24 microg/mL and contained the expected heavy and light chain fragments and N-linked glycan structures. Both AEVZ 5.1 and AVEZ 4 antibodies were IgG1 and recognized the viral coat protein glycoprotein E; both showed complement-independent and complement-enhanced neutralization. Changing the isotype of AEVZ 5.1 from IgG1 to IgG3 (AEVZ 5.3) further enhanced VZV neutralization in the presence of complement, but reduced its neutralization capacity in the absence of complement. Complement enhancement was consistent with our findings that the IgG3 form could bind more molecules of C1q. The results demonstrate the successful use of recombinant methods to generate stable, functional monoclonal antibodies. Modifications of the original antibodies were made with the aim of improving functionality. The resulting cell lines could be used for large-scale production of well-characterized antibodies for therapeutic use.     

Hypogalactosylation of serum IgG in patients with ANCA-associated systemic vasculitis

The triad of small vessel vasculitides (SVV) comprise Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and Churg-Strauss syndrome (CS). All three are associated with presence of circulating IgG antineutrophil cytoplasm antibodies (ANCA) which target autoantigens contained, primarily, within neutrophil azurophilic granules. The widely accepted model of pathogenesis suggests that ANCA activate cytokine-primed neutrophils within the microvasculature, leading to by-stander damage to endothelial cells, and rapid escalation of inflammation with recruitment of mononuclear cells. Activation may be initiated, in vitro, by the coligation of the PR3 or MPO antigen, translocated to the cell surface, and FcgammaRIIa/FcgammaRIIIb receptors. This suggests that the IgG subclass profile of ANCA and, possibly, its glycosylation status could influence the inflammatory mechanisms activated. The glycosylation status of total IgG isolated from the sera of patients with WG (13), MPA (6) and CSS (1) was determined by analysis of the released oligosaccharides. A deficit in IgG galactosylation is demonstrated for all patient samples, compared to controls. The mean percentage values for the agalactosylated (G0) oligosaccharides were 57% (SD +/- 9.71), 47% (SD +/- 4.25) and 28% (SD +/- 4.09) for WG, MPO and control samples, respectively. The G0 levels for polyclonal IgG isolated from the sera of both WG and MPA patients were significantly increased compared to controls (P < 0.0001). The major glycoform present therefore is agalactosylated (G0) IgG. In previous studies the G0 glycoform of IgG has been shown to bind and activate mannan binding lectin, and hence to activate the complement cascade, and to facilitate mannose receptor binding and the uptake of IgG complexes by macrophages and dendritic cells. Both of these activities could impact on the processing and presentation of self-antigens in autoimmune disease.     

A human monoclonal IgG1 potently neutralizing the pro-inflammatory cytokine GM-CSF

The pro-inflammatory cytokine GM-CSF is aberrantly produced in many autoimmune and chronic inflammatory human diseases. GM-CSF neutralization by antibodies has been shown to have a profound therapeutic effect in animal models of rheumatoid arthritis, inflammatory lung diseases, psoriasis and multiple sclerosis. Moreover, the absence of GM-CSF in null mutant mice ameliorates or prevents certain of these diseases. Here we describe the biophysical and biological properties of a human anti-GM-CSF IgG1 antibody designated MT203, which was derived by phage display guided selection. MT203 bound with picomolar affinity to an epitope on human and macaque GM-CSF involved in high-affinity receptor interaction. As a consequence, the antibody potently prevented both GM-CSF-induced proliferation of TF-1 cells with a sub-nanomolar IC50 value and the production of the chemokine IL-8 by U937 cells. MT203 neutralized equally well recombinant (r) human (h) GM-CSF from Escherichia coli and yeast, and also normally glycosylated GM-CSF secreted by human lung epithelial cells in response to IL-1beta stimulation. Furthermore, MT203 significantly reduced both survival and activation of peripheral human eosinophils as may be required for effective treatment of inflammatory lung diseases. The antibody did not show a detectable loss of neutralizing activity after 5 days in human serum at 37 degrees C. Based on its favorable properties, MT203 has been selected for development as a novel anti-inflammatory human monoclonal antibody with therapeutic potential in a multitude of human autoimmune and inflammatory diseases.     

Vaccination of mice with gonococcal TbpB expressed in vivo from Venezuelan equine encephalitis viral replicon particles

We investigated the immunogenicity of gonococcal transferrin binding protein B (TbpB) expressed with and without a eukaryotic secretion signal from a nonpropagating Venezuelan equine encephalitis virus replicon particle (VRP) delivery system. TbpB was successfully expressed in baby hamster kidney (BHK) cells, and the presence of the eukaryotic secretion signal not only apparently increased the protein's expression but also allowed for extracellular localization and glycosylation. Mice immunized with VRPs produced significant amounts of serum antibody although less than the amounts produced by mice immunized with recombinant protein. The response of mice immunized with VRPs encoding TbpB was consistently more Th1 biased than the response of mice immunized with recombinant protein alone. Boosting with recombinant protein following immunization with TbpB VRPs resulted in higher specific-antibody levels without altering the Th1/Th2 bias. Most of the immunization groups produced significant specific antibody binding to the intact surface of the homologous Neisseria gonorrhoeae strain. Immunization with TbpB VRPs without a eukaryotic secretion signal generated no measurable specific antibodies on the genital mucosal surface, but inclusion of a eukaryotic secretion signal or boosting with recombinant protein resulted in specific immunoglobulin G (IgG) and IgA in mucosal secretions after TbpB VRP immunization. The TbpB VRP system has potential for an N. gonorrhoeae vaccine.     

Zonal variation in the distribution of an alpha 1-acid glycoprotein glycoform receptor in human adrenal cortex

Using a histochemical technique with three different alpha 1-acid glycoprotein glycoform one glycoform specific receptor has been identified in human adrenal cortex. The receptor is associated to alpha 1-acid glycoprotein glycoform B and alpha 1-acid glycoprotein glycoform C. The glycoform specific receptor was located in the cytoplasm of glomerulosa and outer fasciculata cells. The intensity of the reaction product decreased in the fasciculata, and no staining was seen in inner fasciculata and reticularis. Inhibition with the simple sugars, mannose and GlcNAc confirmed a lectin-like reaction. The binding activity was dependent on the presence of calcium ions and not on thiol reagents. Thus the lectin-like receptor may belong to the C-type lectin family. Using an antibody to alpha 1-acid glycoprotein the presence of alpha 1-acid glycoprotein was observed in the same location as the glycoform specific receptor. The binding of alpha 1-acid glycoprotein glycoform B and alpha 1-acid glycoprotein glycoform C to the glycoform specific receptor is inhibited by the steroid hormones cortisone, aldosterone, estradiol and progesterone but not by testosterone. The pronounced changes in the distribution of AGP and its glycoform receptors during cell differentiation in the adrenal cortex suggest that AGP and its complementary lectins belong to the group of lectins which control differentiation and spatial position.     

IFN-gamma-independent IgG2a production in mice infected with viruses and parasites

After infection with some viruses and intracellular parasites, antibody production is restricted to IgG2a. We first observed that, whereas live viruses such as lactate dehydrogenase-elevating virus (LDV) or mouse adenovirus induced mostly an IgG2a response, a large proportion of antibodies produced against killed viruses were IgG1. This IgG1 antiviral response was suppressed when live virions were added to inactivated viral particles. These results indicate that the IgG2a preponderance is related to the infectious process itself rather than to the type of antigen involved. Since IFN-gamma is known to stimulate IgG2a production by activated B lymphocytes and to be secreted after infection, we examined the role of this cytokine in the antibody isotypic distribution caused by LDV. Most IgG2a responses were relatively unaffected in mice deficient for the IFN-gamma receptor or treated with anti-IFN-gamma antibody. A similar IFN-gamma-independent IgG2a secretion was observed after infection with the parasites Toxoplasma gondii and Trypanosoma cruzi. However, the IFN-gamma-independent IgG2a production triggered by infection still required the presence of functional T(h) lymphocytes. Therefore, signal(s) other than IFN-gamma secretion may explain the T(h)-dependent isotypic bias in antibody secretion triggered by viruses and parasites.     

The Thomsen-Friedenreich Antigen and αGal-specific Human IgG Glycoforms: Concanavalin A Reactivity and Relation to Survival of Cancer Patients

Glycan structures of IgG strongly influence the affinity for Fcγ receptors and antibody effector functions. However, no particular attention has been paid yet to the glycosylation of tumor antigen-specific IgG. The objectives of this study were (i) to investigate the concanavalin A lectin (ConA) reactivity of human anti-Thomsen-Friedenreich (TF) and anti-αGal specific IgG in gastric cancer patients and healthy controls and (ii) to evaluate whether the ConA-reactivity of anti-TF and anti-αGal specific IgG is associated with the survival rate of patients with cancer. Total IgG was purified from the sera of patients with gastric cancer and healthy blood donors. The anti-TF and anti-αGal glycotope specific IgG were detected with ELISA using synthetic saccharide-polyacrylamide conjugates as antigen. In parallel plate, the ConA reactivity of the anti-TF or anti-αGal IgG was determined and the ConA index was calculated. Results show that serum anti-TF specific IgG antibodies of patients with cancer contain significantly higher content of ConA positive IgG glycoform compared to IgG of controls. No correlation between the ConA reactivity of anti-TF IgG and anti-αGal IgG was observed. High level of anti-TF IgG ConA reactivity was associated with a significantly lower survival rate of patients with gastric cancer.     

Influence of plant lipids on immune responses in mice to the major Brazil nut allergen Ber e 1

BACKGROUND: Lipids, particularly bacterial lipopolysaccharide, can impact on immune responses to proteins, with low doses enhancing type 2 responses. OBJECTIVE: We have examined the influence of natural plant lipid extracts on antibody responses provoked in mice by recombinant Ber e 1, the major allergen in Brazil nuts. METHODS: BALB/c strain mice were immunized (by intraperitoneal injection) with natural or recombinant Ber e l produced in Pichia pastoris and admixed with various lipid fractions isolated from Brazil nuts. Serum samples were analysed for specific IgE antibody by homologous passive cutaneous anaphylaxis assay and for IgG by enzyme-linked immunosorbant assay. RESULTS: Exposure to recombinant (lipid-free) Ber e 1 alone failed to induce detectable IgG or IgE antibody. Co-administration of the total lipid fraction (with reduced triglyceride levels), sterol-rich, or polar lipid fractions, resulted in marked adjuvant effects on IgG and IgE. However, the beta-sitosterol and glycolipid-rich fractions were associated with only low-level IgG antibody, and had little impact on IgE antibody production. Natural Ber e 1 containing endogenous lipids also provoked IgG and IgE antibody responses. Identical IgE and IgG antibody responses were detected regardless of whether natural or recombinant Ber e 1 was used as substrates for analyses. CONCLUSION: Endogenous Brazil nut lipids are required for the induction of optimal antibody responses to Ber e 1 in the BALB/c strain mouse. Appropriate antibody binding sites are present on both natural and recombinant forms of Ber e 1, suggesting that the impact of lipid is at the induction phase, rather than antibody recognition, and is possibly required for efficient antigen presentation.     

Substitution of Pichia pastoris-derived recombinant proteins with mannose containing O- and N-linked glycans decreases specificity of diagnostic tests

BACKGROUND: Recombinant proteins from Pichia pastoris need to be fully evaluated before used as diagnostic tools. OBJECTIVE: The objective of this study was to investigate whether glycosylation by P. pastoris interferes with the specificity of diagnostic tests. METHODS: An autoantigen involved in Wegener's disease (protease 3) and 2 major inhalant allergens from grass pollen (Dac g 5) and house dust mite (Der p 1) were produced as recombinant molecules in P. pastoris. O-linked glycans on Dac g 5 were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The immune reactivity of the recombinant proteins was compared to that of their natural counterparts by ELISA and a radio-allergosorbent test (RAST) as well as by ELISA and RAST inhibition. Results: In contrast to the non-glycosylated natural allergen, recombinant Dac g 5 was shown to carry at least 2 small mannose-containing O-glycans. We showed that both these O-glycans and the N-linked glycans on recombinant protease 3 and recombinant Der p 1 were recognized in ELISA by IgG antibodies in sera of healthy individuals. These IgG responses were closely correlated. The natural autoantigen and allergens were not recognized by IgG antibodies from healthy subjects. The carbohydrate nature of the epitopes recognized by IgG on the recombinant proteins was confirmed by inhibition studies with mannose and yeast mannan. IgE recognition of yeast glycans was observed in 2 out of 9 positive sera from patients with allergic bronchopulmonary aspergillosis. CONCLUSION: Production of recombinant molecules in yeast (or moulds) can introduce IgG-binding glycans that negatively affect the specificity of diagnostic tests.