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Carbohydrate Interactions and HIV-1
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Cytotoxic T cells (CTL) recognize short peptide epitopes presented by class I glycoproteins encoded by the major histocompatibility complex (MHC). It is not yet known whether peptides containing posttranslationally modified amino acids can also be recognized by CTL. To address this issue, we have studied the immunogenicity and recognition of a glycopeptide carrying an O-linked N-acetylglucosamine (GlcNAc) monosaccharide-substituted serine residue. This posttranslational modification is catalyzed by a recently described cytosolic glycosyltransferase. We show that glycosylation does not affect peptide binding to MHC class I and that glycopeptides can elicit a strong CTL response that is glycopeptide specific. Furthermore, glycopeptide recognition by cytotoxic T cells is dependent on the chemical structure of the glycan as well as its position within the peptide.
In the present study we investigated to what extent the peripheral carbohydrate structure of N-linked glycans influences the antigenic properties of human immunodeficiency virus type 1 glycoprotein 120 (gp120). Recombinant gp120 was purified from GMK cells infected with a recombinant vaccinia virus expressing gp120. Purified gp120 was then coated onto 96-well ELISA microplates and subjected to sequential removal of peripheral monosaccharide units. Modified or unmodified gp120 was then incubated with monoclonal antibodies recognizing specific epitopes of gp120 and with a reporter lectin to determine the extent of carbohydrate elimination. Antibody and lectin binding was quantified in an enzyme-linked system. We found that the carbohydrate structure NeuAc-Gal beta (1-4) of N-linked glycans, defined both by lectin reactivity and by specific glycosidases, is involved in modulating the binding of antibody to a number of epitopes of peptide nature. The binding of antibody to one class of epitopes, situated in a region between amino acids 200 and 230, was strongly increased by removal of NeuAc-Gal beta (1-4), whereas the binding to epitopes in the V3 region was decreased and the binding to epitopes in the far N-terminal region was not altered by the treatment. These results suggested that peripheral structures of N-glycans are involved in modulating the overall conformation of gp120.
The lectin concanavalin A (Con A) binds methyl alpha-D-mannopyranoside (Me alpha Man) as well as alpha-D-mannosyl groups at the nonreducing terminus of oligosaccharides. Ligand peptides that mimic the binding of Me alpha Man to Con A were identified from screening an epitope library composed of filamentous phage displaying random hexapeptides. A consensus sequence was identified among affinity-purified phage; Con A binds phage bearing this sequence and is inhibited from doing so by Me alpha Man. When tested for binding against a panel of lectins, phage bearing this sequence bind only weakly to a closely related D-mannose-binding lectin, indicating that binding to Con A is highly selective. A synthetic peptide bearing the consensus sequence blocks the precipitation of Con A by dextran with an inhibition strength equivalent to that of methyl alpha-D-glucopyranoside. These results demonstrate that the specificity of Con A is not limited to carbohydrates and that highly selective sugar-mimics for lectins of plant, animal, or bacterial origin may be identified from epitope libraries.
The cancer-related mucin-type carbohydrate neoantigen Tn was found on gp160 and gp120 of human immunodeficiency virus (HIV). Immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) against Tn neutralized infection with cell-free virus and blocked fusion between HIV-infected and uninfected cells. This inhibition was found in infection of both lymphocytic cells and monocytoid cells. Viruses tested included six HIV-1 and five HIV-2 isolates propagated in different cells, as well as infectious plasma from AIDS patients. The antiviral effect of anti-Tn MAbs occurred by specific binding of the MAb to the virus; this binding was inhibitable by pure Tn antigen, and indications were found that this inhibition occurred at a pre-entry step. Boosting the naturally occurring low-titer anti-Tn activity may be of prophylactic value, as suggested by the in vitro neutralization found in this study.
A panel of anti-gp120 human monoclonal antibodies (HuMAbs), CD4-IgG, and sera from people infected with human immunodeficiency virus type 1 (HIV-1) was tested for neutralization of nine primary HIV-1 isolates, one molecularly cloned primary strain (JR-CSF), and two strains (IIIB and MN) adapted for growth in transformed T-cell lines. All the viruses were grown in mitogen-stimulated peripheral blood mononuclear cells and were tested for their ability to infect these cells in the presence and absence of the reagents mentioned above. In general, the primary isolates were relatively resistant to neutralization by the MAbs tested, compared with the T-cell line-adapted strains. However, one HuMAb, IgG1b12, was able to neutralize most of the primary isolates at concentrations of < or = 1 microgram/ml. Usually, the inability of a HuMAb to neutralize a primary isolate was not due merely to the absence of the antibody epitope from the virus; the majority of the HuMAbs bound with high affinity to monomeric gp120 molecules derived from various strains but neutralized the viruses inefficiently. We infer therefore that the mechanism of resistance of primary isolates to most neutralizing antibodies is complex, and we suggest that it involves an inaccessibility of antibody binding sites in the context of the native glycoprotein complex on the virion. Such a mechanism would parallel that which was previously postulated for soluble CD4 resistance. We conclude that studies of HIV-1 neutralization that rely on strains adapted to growth in transformed T-cell lines yield the misleading impression that HIV-1 is readily neutralized. The more relevant primary HIV-1 isolates are relatively resistant to neutralization, although these isolates can be potently neutralized by a subset of human polyclonal or monoclonal antibodies.
The major target of the neutralizing antibody response to infection by the human immunodeficiency virus type 1 (HIV-1) is the outer envelope glycoprotein, gp120. The spectrum of HIV-1 neutralization specificity is currently represented by monoclonal antibodies (mAbs) that can be divided broadly into five groups. We have studied the binding of these mAbs to functional oligomeric and soluble monomeric gp120 derived from the molecular clone of a cell line-adapted isolate of HIV-1, and compared these binding properties with virus neutralization. Binding of all mAbs except those reactive with the V3 loop was much weaker to oligomeric than to monomeric gp120. This reduction in binding to oligomeric gp120 was determined mostly by a slower relative rate of association, although the dissociation rate also had some influence on relative variation in mAb affinity. Virus neutralization correlated broadly with mAb binding to the oligomeric rather than to the monomeric form of gp120, and neutralization potency was related to the estimated association rate. Thus, with the exception of the hypervariable V3 loop, regions of HIV-1 gp120 with the potential to induce a neutralization response are likely to be poorly presented for antibody recognition on the surface of cell line-adapted virions.
Primary human immunodeficiency virus type 1 (HIV-1) isolates were obtained from 22 patients with AIDS from northern Thailand, where HIV-1 is transmitted primarily through the heterosexual route. Viral sequences were determined for the 22 patients with AIDS, and all were subtype E HIV-1 on the basis of sequence analysis of a region from the envelope protein gp120. Syncytium-inducing (SI) viruses were detected for 16 of 22 patients with AIDS by using MT-2 cells. Characteristics of amino acid sequences in V3 which have not been reported previously for subtype B SI HIV-1 were associated with the subtype E HIV-1 SI phenotype. The SI viruses from our study population contain predominantly a GPGR or GPGH motif at the tip of the V3 loop, in contrast to the previously described subtype E HIV-1 from Thailand which contained predominantly GPGQ. All the SI viruses lost a potential N-linked glycosylation site in V3 which is highly conserved among previously described subtype E HIV-1 isolates from asymptomatic patients from Thailand. HIV-1 envelope sequences including V3 from some patients with AIDS were significantly more divergent than viruses from asymptomatic patients in Thailand characterized 2 years ago or earlier. These results suggest that emergence of subtype E SI HIV-1 variants is associated with the development of AIDS, as it is for subtype B HIV-1. The divergence of subtype E HIV-1 in patients with AIDS as the disease progresses, and the divergence of subtype E HIV-1 in the infected population as the epidemic continues in Thailand, may have important implications for vaccine development.
In this review we have attempted to define the characteristics of TI-2 antigens that enable them to stimulate antibody production in the absence of T cell help. One of the most critical properties of this group of antigens is their ability to deliver prolonged and persistent signaling to the B cell. This by itself is not however sufficient to stimulate Ig synthesis, and they must therefore stimulate non-T cells to interact with the B cells either directly or indirectly via cytokine production. There is evidence implicating the NK cell and T cell as playing this important role in response to TI antigens. Furthermore, we discuss the importance of cytokines such as IL-3, GMCSF, and IFN-gamma, which significantly enhance antibody production by these antigens. Finally, we present evidence demonstrating that B cell activation via TI stimuli does not play merely a permissive role in allowing for cell cycle entry and enhanced responsiveness to other stimuli. Rather, the nature of the B cell activating signal is critical in determining the quantitative and qualitative profile of Ig isotype production.
The paradox that group-specific neutralizing antibodies (NA) exist in the majority of human immunodeficiency virus type 1 (HIV-1)-infected patients, whereas the NA response against autologous HIV-1 virus isolates is highly type-specific, motivated us to study the type- and group-specific NA responses generated upon presentation of escape virus, and the viral epitopes involved in the escape. Patients with demonstrable escape virus all developed group-specific NA, which were detectable after a delay and disappeared prior to disease development. The sera tested inhibited the binding of recombinant soluble gp120IIIB to cell-associated CD4, but group-specific virus neutralization required binding of NA to HIV-1 prior to viral attachment to target cells. Consecutive escape virus isolates were tested for sensitivity to neutralization by heterologous sera. Only minor differences were demonstrated, suggesting that the majority of the change in neutralization sensitivity is driven by the selective pressure of type-specific NA. Furthermore, no differences were observed in sensitivity to neutralization by anti-carbohydrate neutralizing monoclonal antibodies or the lectin concanavalin A, indicating a conserved nature of certain carbohydrate neutralization epitopes during escape. Finally the V3 sequence of three sets of consecutive virus isolates were analysed revealing amino acid mutations in V3 sequences of all escape virus isolates. The biological significance of these variations was confirmed further by the demonstration of changes in sensitivity to neutralization by anti-V3 monoclonal antibodies. These results strongly suggest a participation of the NA response against the V3 loop in the immunoselection of escape virus.
Sequence analysis of the variable regions of the heavy and light chains of the anti-idiotypic antibody 6F9, which mimics the meningococcal group C capsular polysaccharide (MCP), was performed. The immunogenic site on 6F9 responsible for inducing an anti-MCP antibody response was determined by means of sequence and computer model analysis of these data. Complementarity-determining region 3 (CDR3) was found to be unique in that the sequence tract YRY was exposed on the surface. A synthetic peptide spanning the CDR3 domain was synthesized and complexed to proteosomes (meningococcal group B outer membrane protein). Immunizations of BALB/c mice with the peptide-proteosome complex resulted in a significant anti-MCP antibody response. Immunized mice were protected against infection with a lethal dose of Neisseria meningitidis serogroup C.
Immunodeficiency caused by HIV infection probably results from profound dysregulation of normal T lymphocyte properties by the virus. Despite description of the virus cytopathicity and numerous modifications in T cell functions, such as perturbation of antigen receptor signaling, CD4 downregulation, and induction of apoptosis, the precise mechanisms underlying the disruption of normal immune responses have not yet been elucidated. In the present study, we show that HIV-1-infected lymphocytes of the CEM cell line (either latent or virus-producing) and HIV-1-infected CD4+ lymphocytes have several membrane proteins with altered glycosylation patterns. Using lectins with specificity for different carbohydrate moieties, we could demonstrate the presence of two exposed nonsialylated disaccharides: a terminal Gal beta 1-->3GalNAc and a terminal Gal beta 1-->4GlcNAc. In particular, CD45, one of the major T cell glycoproteins, appeared to be partially sialylated on N- and O-linked carbohydrate moieties. Concerning the latter, PNA lectin which recognizes nonsialylated terminal Gal beta 1-->3GalNAc might precipitate up to 75% of the total tyrosine phosphatase activity displayed by CD45 molecules from one latently HIV-1-infected CEM cell line. Since CD45 glycoproteins are thought to play an important regulatory role in cell-to-cell interactions owing to their variable extracellular region and because they may regulate membrane signaling through their intracellular phosphatase domains, we suggest that these altered CD45 molecules may present an abnormal signal for natural ligands such as the B-cell-specific surface receptor CD22, thus perturbing the normal immune response in HIV-1-infected individuals.
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been shown to be extensively modified by N-linked glycosylation; however, the presence of O-linked carbohydrates on the glycoprotein has not been firmly established. We have found that enzymatic deglycosylation of the HIV-1 envelope glycoprotein with neuraminidase and O-glycosidase results in a decrease in the apparent molecular weight of the envelope glycoprotein. This result was observed in both vaccinia virus recombinant-derived envelope glycoproteins and glycoproteins derived from the IIIB, SG3, and HXB2, strains of HIV-1. The decrease in molecular weight was also observed when the envelope glycoprotein had been deglycosylated with N-glycanase F after treatment with neuraminidase and O-glycosidase, indicating that the decrease in apparent molecular weight was not attributable to the removal of N-linked carbohydrate. Treatment with neuraminidase, O-glycosidase, and N-glycanase F was found to be necessary to remove all radiolabel from [3H]glucosamine-labelled envelope glycoprotein, a result seen for both recombinant and HIV-1-derived envelope glycoprotein. [3H]glucosamine-labelled carbohydrates liberated by O-glycosidase treatment were separated by paper chromatography and were found to be of a size consistent with O-linked oligosaccharides. We, therefore, conclude that the HIV-1 envelope glycoprotein is modified by the addition of O-linked carbohydrates.
Human immunodeficiency virus (HIV) virion RNA and proviral DNA sequences have been examined over a 1-year period in an HIV-seropositive patient, commencing with the start of zidovudine treatment. By characterizing the variable V3 and V4 env domains, four related but structurally discrete genotypes could be identified prior to the start of therapy and during the subsequent 60-week period of therapy. Each of the four subtypes showed a unique pattern in the preservation of glycosylation sites. A comparison of the V3 amino acid sequences in peripheral blood mononuclear cell proviral DNA and plasma virion RNA at 0, 24, 36, and 60 weeks demonstrated that proviral DNA did not serve as a predictor of the structure of virion RNA. HIV virion RNA subtype 3 was the most prevalent virion RNA subtype at three of the four periods studied, yet no corresponding proviral DNA was detected. Other virion subtypes have been observed, but only on a transient basis. The present data are consistent with a model of HIV infection in which related but different HIV substrains coexist and evolve independently within an individual. Characterization of virion RNA may be required to identify the unique properties of the virus involved in disease progression; characterization of proviral DNA will not yield this information.
We discovered recently that a subset of mouse anti-streptococcal mAbs cross-reacted with N-acetyl-beta-D-glucosamine (GlcNAc) and certain cytoskeletal proteins, and recognized both carbohydrate and peptide antigenic determinants. To further study the nature and biologic significance of immunologic mimicry between carbohydrate and peptide Ags, eight human hybridomas secreting anti-GlcNAc mAbs were produced by in vitro stimulation of PBL with streptococcal peptidoglycan-polysaccharide complexes and pokeweed mitogen. All human anti-GlcNAc mAbs described in this study were shown to express marked cross-reactivity with keratin from human skin in the ELISA and Western immunoblot. Mapping of the mAbs with overlapping synthetic decapeptides of the entire amino acid sequence of human cytokeratin 14 revealed that human anti-GlcNAc mAbs recognized specific cytokeratin decapeptides. Four human anti-GlcNAc mAbs recognized a single cytokeratin decapeptide whereas two mAbs reacted with several individual peptide epitopes in different fragments of cytokeratin 14. In addition, two mAbs, 1.C8 and 9.B12, reacted with multiple cytokeratin decapeptides, predominantly in the head domain of the molecule, and their reactivity correlated with positive binding of the mAbs to cytokeratin 14 in the Western immunoblot and with positive staining of human epidermis in the indirect immunofluorescent assay. Finally, we demonstrated that Abs to keratin and synthetic keratin decapeptides were induced in BALB/c mice immunized with GlcNAc-BSA but not with BSA, suggesting that the anti-keratin Ab response in vivo may be driven by nonkeratin Ags containing terminal O-linked GlcNAc.
Tumour development is usually associated with changes in cell surface carbohydrates. These are often divided into changes related to terminal carbohydrate structures, which include incomplete synthesis and modification of normally existing carbohydrates, and changes in the carbohydrate core structure. The latter includes chain elongation of both glycolipids and proteins, increased branching of carbohydrates in N‐linked glycoproteins, and blocked synthesis of carbohydrates in O‐linked mucin‐like glycoproteins. In mature organisms, expression of distinct carbohydrates is restricted to specific cell types; within a given tissue, variation in expression may be related to cell maturation. Tumour‐associated carbohydrate structures often reflect a certain stage of cellular development; most of these moieties are structures normally found in other adult or embryonic tissues. There is no unique tumour carbohydrate structure, since certain structures which are tumour‐related in one organ may be normal constituents of other tissues. Tumour‐associated carbohydrate changes have been used in the diagnosis of human cancers. Recently, however, it has been demonstrated that the expression of some carbohydrate structures is associated with prognosis. Tn, sialyl‐Tn, and T are cell membrane‐bound mucin‐like carbohydrate structures that may be expressed in tumours due to blocked synthesis of the core carbohydrate chain of mucin‐like structures. Their expression is strongly associated with prognosis in certain tumours, but the biological relationship between their expression and tumour progression is at present unknown. The blood group‐related carbohydrate structures Lex, sialyl‐Lex, ABH, and Ley are examples of terminal carbohydrate structures which are related to tumour prognosis. These structures are of increasing interest since they may function as adhesion molecules; adhesion of tumour cells to endothelial cells of blood vessels may be mediated by an interaction between sialosyl‐Lex and E‐selectin and studies indicate that Ley is related to cell motility. These findings are now the basis for tumour therapeutic experiments.
Ley determinant (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----R) defined by mAb BM-1 is highly expressed in human immunodeficiency virus (HIV)-infected T cell lines and in CD3+ peripheral mature T cells of patients with acquired immune deficiency syndrome (AIDS) or with AIDS-related complex (ARC). Ley expression increased greatly in the CD3+ population in the advanced stage of AIDS when the CD4+ population decreased greatly. Six other carbohydrate antigens tested by their respective mAbs were not detected in these same cells. None of the carbohydrate antigens tested by the seven mAbs used in this study were found in noninfected T cell lines and in normal peripheral blood lymphocytes.
The primary molecular changes that lead to development of acquired immunodeficiency syndrome (AIDS) are very poorly understood, as are the mechanisms underlying the protection of the developing human from the maternal immune response. Recent data suggests that the human immunodeficiency virus (HIV) may be using the glycosylation system of the T lymphocytes to acquire glycans for its glycoproteins that enable it to disrupt carbohydrate dependent immune cell interactions or induce aberrant immune reactions. Consistent with this hypothesis, gp120 from HIV infected human H9 lymphoblastoid cells expresses biantennary N-linked glycans with a bisecting GlcNAc sequence on 11% of their total oligosaccharides. This specific carbohydrate sequence has recently been shown to protect K562 erythroleukemic cells from natural killer (NK) cell responses when presented on the cell surface. We have recently demonstrated that bisecting biantennary type N-linked glycans are also expressed on the human zona pellucida (ZP); previous lectin binding studies indicate that it is also expressed on human spermatozoa. Thus both the human gametes and HIV produced by H9 cells carry this same protective carbohydrate epitope on their outer surfaces. Human afetoprotein expressed in the developing human also carries the bisecting GlcNAc sequence, indicating that it may be suppressing the emerging fetal immune response by using its carbohydrate sequence as a functional group. We have suggested that the developing human and the gametes are also protected by soluble immunosuppressive glycoproteins found in the amniotic fluid and seminal plasma known as glycodelin-A (GdA) and glycodelin-S (GdS) respectively. Structural analysis of their N-linked oligosaccharides combined with other functional studies suggest that GdA and GdS employ their very unusual carbohydrate sequences as functional groups that enable them to manifest their immunosuppressive activities. GdA and GdS are significant components of our recently proposed model for the protection of the developing human and gametes designated the human fetoembryonic defence system hypothesis. A striking relationship now emerging is that the same unusual carbohydrate sequences associated with these immunosuppressive glycodelins are also specifically expressed on intravascular helminthic parasites, Helicobacter pylori, human tumour cells, and HIV infected T lymphocytes. The information presented in this review suggests that two new corollaries should be added to our recently proposed defence system hypothesis: (i) mimicry or acquisition of glycans that are used in this protective system by pathogens or tumour cells may enable them to either subvert or misdirect the human immune response, thereby greatly increasing their pathogenicity; and (ii) expression of glycoconjugates used in this system by normal cells and tissues outside the reproductive system may protect them from immune responses, especially in those cases where major histocompatibility recognition is either absent or minimal. A better understanding of this hypothesis and its corollaries may enable us to address the molecular mechanisms underlying not only AIDS but also a host of other very serious pathological conditions in the human.
We have previously demonstrated that human immunodeficiency virus (HIV) envelope glycoproteins have specific carbohydrate-binding properties for mannosyl/N-acetylglucosaminyl residues presented at high density on a carrier in vitro. Here, we investigated whether HIV envelope glycoprotein gp120 was able to interact with surface membrane carbohydrates of CD4(+) cells by means of such lectin-carbohydrate interactions, CD4-free tryptic glycopeptides, prepared from the membrane of CD4(+) monocytic U937 cells and partially purified by ConA-agarose affinity chromatography, could be eluted by mannan but not by methyl-alpha-mannose or methyl-alpha-glucose, which strongly suggests that they displayed oligomannosidic structures, These glycopeptides bound in a mannosyl-specific manner to radiolabeled recombinant gp120, Deglycosylation with N-glycanase which, as expected, strongly diminished binding of the glycopeptides to concanavalin A also abolished their interaction with gp120, In addition, the glycopeptides inhibited HIV infection of both U937 and CD4(+) lymphoid CEM cells when preincubated with the virus, These findings indicate that, independently of the binding to CD4, mannosyl structures on CD4(+) sells may play a role through lectin-carbohydrate interactions in envelope glycoprotein binding to a putative coreceptor(s) of HIV.
CD43 (leukosialin, gpL115, sialophorin) is a major sialoglycoprotein widely expressed on hematopoietic cells that is defective in the congenital immunodeficiency Wiskott-Aldrich syndrome. It is thought to play an important role in cell-cell interactions and to be a costimulatory molecule for T lymphocyte activation. Using a metabolic 35SO4(2-) radiolabeling assay or biotinylation of cell surface proteins, we describe here that CD43 are sulfated molecules the glycosylation of which is altered in human immunodeficiency virus type 1 (HIV-1)-infected leukemic T cells of the CEM line. Hyposialylation of O-glycans and changed substitution on N-acetylgalactosamine residues are observed. The glycosylation defect is associated with an impairment of CD43-mediated homotypic aggregation which can be restored by resialylation. The hyposialylation of CD43 on HIV-1+ cells may explain the high prevalence of autoantibodies directed against nonsialylated CD43 that have been detected in HIV-1-infected individuals. A defect in glycosylation of important molecules such as CD43 or, as we recently described, CD45 may explain alterations of T cell functions and viability in HIV-1-infected individuals. In addition, a possible implication of hyposialylation in the HIV-1-infected cells entrapment in lymph nodes could be envisioned.
Tumour development is usually associated with changes in cell surface carbohydrates. These are often divided into changes related to terminal carbohydrate structures, which include incomplete synthesis and modification of normally existing carbohydrates, and changes in the carbohydrate core structure. The latter includes chain elongation of both glycolipids and proteins, increased branching of carbohydrates in N-linked glycoproteins, and blocked synthesis of carbohydrates in O-linked mucin-like glycoproteins. In mature organisms, expression of distinct carbohydrates is restricted to specific cell types; within a given tissue, variation in expression may be related to cell maturation. Tumour-associated carbohydrate structures often reflect a certain stage of cellular development; most of these moieties are structures normally found in other adult or embryonic tissues. There is no unique tumour carbohydrate structure, since certain structures which are tumour-related in one organ may be normal constituents of other tissues. Tumour-associated carbohydrate changes have been used in the diagnosis of human cancers. Recently, however, it has been demonstrated that the expression of some carbohydrate structures is associated with prognosis. Tn, sialyl-Tn, and T are cell membrane-bound mucin-like carbohydrate structures that may be expressed in tumours due to blocked synthesis of the core carbohydrate chain of mucin-like structures. Their expression is strongly associated with prognosis in certain tumours, but the biological relationship between their expression and tumour progression is at present unknown. The blood group-related carbohydrate structures Lex, sialyl-Lex, ABH, and Ley are examples of terminal carbohydrate structures which are related to tumour prognosis. These structures are of increasing interest since they may function as adhesion molecules; adhesion of tumour cells to endothelial cells of blood vessels may be mediated by an interaction between sialosyl-Lex and E-selectin and studies indicate that Ley is related to cell motility. These findings are now the basis for tumour therapeutic experiments.
The mouse hemoglobin-derived decapeptide Hb (67–76), VITAFNEGLK, which binds well to Ek and is non-immunogenic in CBA/J mice, was O-glycosylated with the tumor-associated carbohydrate Tn (α-D-N-acetylgalactosamine, or α-D-GalNAc). Each of the ten positions in the peptide was substituted with serine or threonine having the Tn antigen attached. The complete set of Tn-glycosylated peptides were then studied for binding to Ek and for immunogenicity in CBA/J mice. All of those glycopeptides which had the Tn attached to serine or threonine at a position in the peptide where, according to the crystal structure determinations, the amino acid side chain was oriented downwards into the binding site of the major histocompatibility complex (MHC) molecule, completely lost their capacity for binding to Ek. This was the case for the glycopeptides with Tn attacted at position 68 and 76, which are the major anchor residues and for those with Tn attached at position 71 and 73, which function as secondary anchor residues. Those glycopeptides which had Tn attached to serine or threonine at positions where the side chain pointed away from the binding site maintained their capacity for binding to Ek, except for those with Tn attached at position 70 and 74. Furthermore, some of the MHC-binding glycopeptides were immunogenic. In particular, this was the case for the glycopeptide with Tn attached to the central position 72 in the decapeptide. From previous studies, this is known to be the dominant T cell receptor contact residue of Hb (67–76). The results suggest that T cells may be capable of recognizing epitopes which are partially defined by a small glycan group.
Abstract The core oligosaccharides of low-molecular-weight lipopolysaccharide (LPS), also termed lipooligosaccharide (LOS), of pathogenic Neisseria spp. mimic the carbohydrate moieties of glycosphingolipids present on human cells. Such mimicry may serve to camouflage the bacterial surface from the host. The LOS component is antigenically and/or chemically identical to lactoneoseries glycosphingolipids and can become sialylated in Neisseria gonorrhoeae when the bacterium is grown in the presence of cytidine 5′-monophospho-N-acetylneuraminic acid, the nucleotide sugar of sialic acid. Strains of Neisseria meningitidis and Haemophilus influenzae also express similarly sialylated LPS. Sialylation of the LOS influences susceptibility to bactericidal antibody, may decrease or prevent phagocytosis, cause down-regulation of complement activation, and decrease adherence to neutrophils and the subsequent oxidative burst response. The core oligosaccharides of LPS of Campylobacter jejuni serotypes which are associated with the development of the neurological disorder, Guillain-Barré syndrome (GBS), exhibit mimicry of gangliosides. Cross-reactive antibodies between C. jejuni LPS and gangliosides are considered to play an important role in GBS pathogenesis. In contrast, the O-chain of a number of Helicobacter pylori strains exhibit mimicry of Lewisx and Lewisy blood group antigens. The role of this mimicry remains to be investigated, but may play a role in bacterial camouflage, the induction of autoimmunity and immune suppression in H. pylori-associated disease.
Recognition of viral Ag and of the envelope glycoprotein of HIV (gp120) in particular by human Th cells is critical in the immune response to the viral Ag which includes antibody production and generation of cytotoxic cells. Procedures to increase antigenicity of gp120 are highly desirable in a vaccine perspective. Therefore, to induce activation of gp120-specific T cells by a liminal dose of Ag we enhanced uptake of gp120 by exploiting the galactose receptors on APC. Terminal sialic acid residues were removed by neuraminidase treatment from the carbohydrate side chains of the heavily glycosylated gp120. Galactose residues were exposed and hence recognized by galactose receptors on APC. The experiments demonstrated that 1) human monocytes and dendritic cells, but not cells of the B lineage, bear galactose receptor; 2) galactose receptors are indeed involved because enhanced presentation is inhibited by galactose and acetylgalactosamine and competed for by other asialoglycoproteins; 3) galactose receptors mediate internalization of Ag in intracellular compartments that intersect the processing and presenting pathways, resulting in activation of specific T cells; 4) antigenicity of gp120 for specific T cells can be enhanced by the exposure of galactose residues.
Previous studies have disagreed about the presence of O-linked carbohydrate epitopes on gp 120 of HIV, although antibodies against short-chain O-linked glycans neutralize HIV infection and block syncytium formation in vitro. To settle this question, we analysed the O-linked glycans of gp 120 by chemical methods using purified HIV-1 gp 120 from cells infected with recombinant vaccinia virus solely expressing gp 160 or gp 120. Alkaline borohydride degradation of recombinant gp 120 released monosaccharides and also slightly larger structures (di/trisaccharides) by a beta-elimination, confirming the presence of simple O-linked oligosaccharides. The functional activity as neutralisation epitopes of the O-linked oligosaccharides expressed on recombinant gp 120 was preserved, since fusion between uninfected CD4+ cells and cells infected with recombinant vaccinia was blocked by monoclonal antibodies to the O-linked oligosaccharides of gp 120. Although the mechanism for HIV induction of O-linked oligosaccharide neoantigens is unknown, these results indicate that the O-linked neutralization epitopes are inherent to the glycoprotein itself, and that the unusual appearance of simple O-linked oligosaccharides on gp 120 is independent of any interaction between the host cell and retroviral genes other than env.
In a retrospective study of 31 pregnant women infected with human immunodeficiency virus type 1 (HIV-1), nine (29%) infants presented unequivocal signs of HIV-1 infection (persistent p24 antigenemia and/or positive virus isolation). All serum samples obtained from the others, during pregnancy and on delivery, were studied for specific antibody (IgA) production by immunoblotting analysis to establish a possible link between the presence of a defined antibody class and mother-to-child viral transmission. The majority (16 of 22) of HIV-1-seropositive mothers who delivered uninfected children showed IgA antibody to low-molecular-weight HIV-1 polypeptides during pregnancy. Among those who delivered infected babies, only one showed a weak IgA reactivity to HIV-1 during pregnancy. Thus, our results suggest that immunoblotting study of IgA may be a diagnostic adjunct to predict the risk of mother-to-child HIV-1 transmission.
Peptide ligands for the carbohydrate-binding protein concanavalin A (Con A) have been identified by screening a large, diverse peptide library expressed on the surface of filamentous phage. A dodecapeptide containing the consensus sequence Tyr-Pro-Tyr was found to bind Con A with an affinity (dissociation constant, Kd) of 46 microM, comparable to that of a known carbohydrate ligand, methyl alpha-D-mannopyranoside (Kd of 89 microM). In addition the peptide inhibited precipitation of the alpha-glucan dextran 1355 by Con A. Given the complexity of oligosaccharide synthesis, the prospect of finding peptides that competitively inhibit carbohydrate-specific receptors may simplify the development of new therapeutic agents.
Three virus isolates HTLV-IIIB/lyA, HTLV-IIIB/lyB and HTLV-IIIB/lyO, obtained by passaging and propagating the HTLV-IIIB/H9 isolate in three separate cultures of mixed peripheral blood mononuclear cells (PBMC) from donors of blood type A, B or O, respectively, were tested for susceptibility for virus neutralization by the monoclonal antibody (MAb) AH16 directed against the blood group A epitope. MAb AH16 was previously shown to inhibit cell-free virus infection using HTLV-IIIB propagated in H9 cells. AH16 showed a concentration-dependent inhibition of the HTLV-IIIB/lyA isolate but did not inhibit the HTLV-IIIB/lyB or the HTLV-IIIB/lyO isolate. Specificity of the MAb-mediated inhibition was shown using A-antigen (tetrasaccharide). Thus, HIV infection of PBMC from donors with blood type A appears to induce expression of host-cell-encoded carbohydrate blood group A epitope on HIV which can be a target for MAb-mediated virus neutralization.
The aim of this study was to determine whether mannosyl-specific lectins, especially Concanavalin A (ConA), may bridge HIV-1 env glycoproteins to cell membranes to increase virus binding to its targets, and to what extent this lectin-carbohydrate interaction can modify HIV-1 infectivity for monocytic compared with lymphoid cells. Monocytic U937 and lymphoid CEM cells, which both express surface mannose, were utilized. Whether first incubated with env glycoprotein or with the cells, lectins bound both to the cells and to radiolabeled recombinant gp160 (rgp160). Thus, they enhanced rgp160 adsorption to the cells in a methyl-alpha-mannose inhibitable manner. ConA did not appear to bind to the V1 domain of CD4 at the U937 cell surface since Leu3a binding was not blocked in the presence of ConA, nor was recombinant CD4 retained on a ConA-agarose affinity matrix. Moreover, enhanced rgp160 binding to the cells was CD4 independent, since it was not modified by preincubating the cells with Leu3a. Finally, ConA did not inhibit the binding of CD4-IgG3 chimeric molecules to virions immobilized on nitrocellulose membrane, which argues against the possibility that it interferes with the interaction of gp120 and CD4. However, both when incubated with the virus or with the cells and despite mediating enhanced binding of env glycoprotein, ConA neutralized HIV-1 infectivity for monocytic U937 as well as for lymphoid CEM cells. In this respect, ConA behaves like neutralizing antibodies which do not interfere with CD4 binding of gp120 but rather with some later event that leads to virus entry. These findings obtained with plant lectins may be of relevance in vivo, inasmuch as endogenous mannosyl-binding proteins, which are known to function as opsonins, have been reported to inhibit in vitro infection by HIV-1.
In this study we raised antibodies against Candida albicans mannans of serotype A and B which comprise mannose alpha(1----2)-and mannose alpha(1----3)-linked residues. These antibodies inhibited human immunodeficiency virus type IIIB (HIV-IIIB) infection of H9 cells in vitro; 5 micrograms/ml of antibodies against mannan from serotype A and 10 micrograms/ml of antibodies against serotype B mannan were sufficient to inhibit infection by almost 100 and 85%, respectively, after an incubation period of 4 days. During a prolonged incubation period (8-12 days), the amount of HIV particles (as measured by reverse transcriptase activity in the culture medium) increased again in assays with antibodies raised against serotype B, but only little in assays containing antibodies against serotype A. Applying the Western blotting technique and a novel enzyme-linked immunosorbent assay system it was established that the antibodies reacted with the gp120 of HIV-1 exclusively. Immunofluorescence inspection using a confocal laser scanning microscope revealed that the gp120 protein is exposed on the outer surface of H9 cells where it is recognized by the anti-mannan antibodies. These results indicate that mannan residues of C. albicans can serve as antigens to raise neutralizing antibodies against HIV infection.
We have probed the structures of monomeric and oligomeric gp120 glycoproteins from the LAI isolate of human immunodeficiency virus type 1 (HIV-1) with a panel of monoclonal antibodies (MAbs); most of these MAbs are directed against continuous epitopes. On native monomeric gp120, most of the first conserved (C1) domain is accessible to MAbs, although some regions of C1 are relatively inaccessible. All of the MAbs directed against the C2, C3, and C5 domains bind preferentially to denatured monomeric gp120, indicating that these regions of gp120 are poorly accessible on the native monomer, although the extreme C terminus in C5 is well exposed. Segments of the V1, V2, and V3 loops are exposed on the surface of monomeric gp120, although the base of the V3 loop is inaccessible. A portion of C4 is also available for MAb binding on monomeric gp120, as is the extreme C terminus in C5. However, on oligomeric gp120-gp41 complexes, only the V2 and V3 loops (and perhaps V1) are well exposed and a segment of the C4 region is partially exposed; continuous epitopes in C1 and C5 that are accessible to antibodies on monomeric gp120 are occluded on the oligomer. Although deletion of the V1, V2, and V3 loops resulted in increased exposure of several discontinuous epitopes overlapping the CD4-binding site, the exposure of most continuous epitopes on the monomeric gp120 glycoprotein was not affected. These results imply a HIV-1 gp120 structure in which the conserved continuous determinants are inaccessible; in some cases, this inaccessibility is due to intramolecular interactions between conserved regions, and in other cases, it is due to intermolecular interactions with other components of the glycoprotein spike. These findings have implications for the design of subunit vaccines based on gp120.
One mechanism for expanding the cellular tropism of human immunodeficiency virus (HIV) in vitro is through formation of phenotypically mixed particles (pseudotypes) with human T lymphotropic virus type I (HTLV-I). In this study we found that pseudotypes allow penetration of HIV particles into CD4-negative cells, previously nonsusceptible to HIV infection. The infection of CD4-negative cells with pseudotypes could be blocked with anti-HTLV-I serum but failed to be significantly inhibited with anti-HIV serum or a V3-neutralizing anti-gp120 monoclonal antibody. This may represent a possibility for pseudotypes to escape neutralization by the immune system in vivo. Previous reports have suggested that carbohydrate structures may be conserved neutralization epitopes on retroviruses. In this study, the neutralizing capacity of lectins and anti-carbohydrate monoclonal antibodies was found to block infection by cell-free pseudotypes in CD4-negative cells. We suggest that although viral cofactors might expand the tropism of HIV in vivo, HIV and HTLV-I seem to induce common carbohydrate neutralization epitopes.
We discovered recently that a subset of mouse anti-streptococcal mAbs cross-reacted with N-acetyl-beta-D-glucosamine (GlcNAc) and certain cytoskeletal proteins, and recognized both carbohydrate and peptide antigenic determinants. To further study the nature and biologic significance of immunologic mimicry between carbohydrate and peptide Ags, eight human hybridomas secreting anti-GlcNAc mAbs were produced by in vitro stimulation of PBL with streptococcal peptidoglycan-polysaccharide complexes and pokeweed mitogen. All human anti-GlcNAc mAbs described in this study were shown to express marked cross-reactivity with keratin from human skin in the ELISA and Western immunoblot. Mapping of the mAbs with overlapping synthetic decapeptides of the entire amino acid sequence of human cytokeratin 14 revealed that human anti-GlcNAc mAbs recognized specific cytokeratin decapeptides. Four human anti-GlcNAc mAbs recognized a single cytokeratin decapeptide whereas two mAbs reacted with several individual peptide epitopes in different fragments of cytokeratin 14. In addition, two mAbs, 1.C8 and 9.B12, reacted with multiple cytokeratin decapeptides, predominantly in the head domain of the molecule, and their reactivity correlated with positive binding of the mAbs to cytokeratin 14 in the Western immunoblot and with positive staining of human epidermis in the indirect immunofluorescent assay. Finally, we demonstrated that Abs to keratin and synthetic keratin decapeptides were induced in BALB/c mice immunized with GlcNAc-BSA but not with BSA, suggesting that the anti-keratin Ab response in vivo may be driven by nonkeratin Ags containing terminal O-linked GlcNAc.
Priming of CD8+ cytotoxic T lymphocyte (CTL) responses with recombinant proteins has been facilitated by the development of novel adjuvants that deliver antigens into the class I major histocompatibility complex (MHC) pathway. However, the extent to which secondary structure or glycosylation of these proteins prevents priming of class I MHC-restricted CTL responses is not clear. To address this issue, recombinant HIV-1 gp120 envelope proteins produced in yeast insect, or mammalian cells were compared for the ability to elicit CD8+ CTL activity in mice. Envelope-specific CD8+ T lymphocytes were detected in BALB/c mice immunized with env 2-3, a 55-kDa yeast-derived envelope protein that is not glycosylated and lacks a native conformation. This response was directed against a previously described epitope in the V3 region of gp120, as well as a newly identified epitope located near the carboxy-terminus of the molecule. Similar levels of V3-directed CTL activity were observed in mice immunized with recombinant gp120 produced in insect (Spodoptera fugiperda) cells using a baculovirus expression system (gp120BAC). In contrast, induction of CTL responses was considerably less efficient when mice were immunized with gp120CHO, a native, fully glycosylated envelope protein produced in mammalian CHO cells. Denaturation of gp120CHO prior to immunization was not sufficient to prime CTL responses. However, envelope-specific CD8+ CTL activity was elicited when N-linked glycans were removed by treatment with an endoglycosidase. Possible mechanisms by which N-linked glycans influence delivery or processing of recombinant proteins for class I MHC presentation, and the implications of these findings for the design of subunit vaccines, are discussed.
Recently, we found that human anti-GlcNAc mAbs reacted with keratin from human skin and recognized specific peptide epitopes of human cytokeratin 14. Our data demonstrate that anti-keratin Ab responses in mice might be driven by Ags bearing terminal O-linked GlcNAc residues. To determine if mouse anti-GlcNAc mAbs recognized peptide epitopes of keratin, several mouse anti-GlcNAc mAbs were reacted with a panel of overlapping synthetic decapeptides of the entire amino acid sequence of human cytokeratin 14 in the ELISA. Results of the ELISA demonstrated that three mAbs, HGAC 54, HGAC 78, and 101.4.1, expressed maximal binding activity to keratin peptide B1 with the amino acid sequence SFGSGFGGGY. In addition, we found that mouse anti-cytokeratin 14 mAb CKB-1 recognized the same peptide and expressed anti-GlcNAc activity as well. Keratin peptide B1 was shown to react not only with anti-GlcNAc mAbs but also with several lectins such as wheat germ agglutinin, Datura stramonium lectin, Lycopersicon esculentum lectin, Solanum tuberosum lectin, and lectin from Wisteria floribunda. Reaction of the lectins with solid-phase keratin peptide B1 was inhibited by soluble GlcNAc and was not inhibited by glucose demonstrating specificity of binding. Using a panel of 24 synthetic keratin B1 peptides, each with a single amino acid substitution, we found that aromatic-aromatic and hydrophobic interactions were the major driving forces in the stabilization of the peptide-protein complexes. Finally, we demonstrated that immunization of BALB/c mice with peptide B1 conjugated to BSA induced an anti-GlcNAc Ab response. Results of our experiments indicated that certain peptides may express functional activity similar to GlcNAc and induce in vivo anti-carbohydrate Ab responses.
Anti-galactocerebroside (GalC) antibodies have been reported to inhibit myelin formation, cause demyelination, and block HIV-I infection of neural cells. We examined the binding of 3 monoclonal and polyclonal anti-GalC antibodies to a panel of purified glycolipids by ELISA and by an immunospot assay on nitrocellulose blots. All 3 antibodies bound strongly to GM1 ganglioside, monogalactosyl diglyceride, and asialo-GM1, and 2 of the antibodies bound to GD1b and psychosine. The anti-GalC antibodies also bound to 3 glycoprotein bands in human neuroblastoma cells on Western blot, and binding to the proteins was abolished by pre-treatment with pronase or with periodate which oxidizes the terminal carbohydrate residues. These results indicate that anti-GalC antibodies cross react with oligosaccharide determinants of other glycolipids and glycoproteins, and that these cross-reactivities may be responsible for some of the biological effects of the anti-GalC antibodies.
Control of pandemic infection of human immunodeficiency virus type 1 (HIV-1) requires some means of developing mucosal immunity against HIV-1 because sexual transmission of the virus occurs mainly through the mucosal tissues. However, there is no evidence as yet that the secretory immunoglobulin A (IgA) antibody induced by immunization with antigens in experimental animals can neutralize HIV-1. We demonstrate here that oral immunization with a new macromolecular peptide antigen and cholera toxin (CT) induces a high titre (1:2") of gut-associated and secretory IgA antibody to HIV-1. Using three different neutralizing assays, we clearly demonstrate that this secretory IgA antibody is able to neutralize HIV-1IIIB, HIV-1SF2 and HIV-1MN. Our new approach may prove to be important in the development of a mucosal vaccine that will provide protection of mucosal surfaces against HIV-1.
The monoclonal antibody (mAb) B3 recognizes an antigen found on the surface of many adenocarcinoma cells. While the structure of the cellular antigen is unknown, epitope mapping using neoglycoproteins with known carbohydrate moieties indicates that the mAb B3 reacts with the LewisY (LeY) antigen [Pastan et al., Cancer Res. 51 (1991) 3781-3787]. We have used mAb B3 to select for peptides that mimic the carbohydrate structure using libraries of filamentous phage displaying random peptides on their surface. Phage that were selected coded for the sequence APWLYGPA. The corresponding peptide was synthesized and tested for its ability to bind to mAb B3. The peptide was found to inhibit specifically the binding of 111In-labeled mAb B3 to A431 adenocarcinoma cells, as well as to inhibit killing of these cells by a B3 immunotoxin. In addition, the LeY carbohydrate, lactodifucotetraose, was able to compete with the phage displaying this peptide for binding to mAb B3. Alanine-scanning mutagenesis of the sequence coding for this peptide indicates that four residues, PWLY, were critical for binding to the mAb. The sequence is similar to other sequences known to mimic carbohydrate structures.
It is well known that antibodies to N-acetyl-beta-D-glucosamine (GlcNAc) cross-react with cardiac valves, skin, and other host tissues. However, molecular targets of these antibodies have not been identified. For this reason, anti-streptococcal mAb cross-reactive with group A streptococci and heart proteins were studied for their reactivity with GlcNAc, the immunodominant epitope of group A streptococcal carbohydrate. Characterization of the mAb that recognized GlcNAc revealed that each mAb had its own unique antigen-binding profile and pattern of immunofluorescence on rat heart cells. In the ELISA and Western blot these mAb reacted with cytoskeletal and heart proteins such as actin, keratin, myosin, and vimentin, as well as with streptococcal recombinant M5 and M6 proteins. Binding of the mAb to cytoskeletal proteins was inhibited by GlcNAc conjugated with BSA in a dose-dependent manner, and the mAb preferentially reacted with high-density GlcNAc-BSA conjugates. Antigenic determinants on the proteins recognized by the mAb were resistant to sodium periodate and N-acetylglucosaminidase treatment, suggesting reactivity with peptide and not carbohydrate structures. On reaction of the mAb with a panel of synthetic streptococcal, viral, and myosin peptides, one of the mAb, 49.8.9, was found to react most strongly with a synthetic peptide sequence synthesized from the coxsackievirus B3 capsid protein VP1, which shows homology with and cross-reacts with sequences in the streptococcal M6 protein and human cardiac myosin. This most interesting mAb, previously shown to neutralize coxsackie viruses, recognized the amino acid sequence RRKLEFF, which may mimic the GlcNAc epitope. The data collected show that we have identified a new group of multireactive autoantibodies that recognize GlcNAc and cytoskeletal proteins, as well as defined peptide epitopes.
Anti-alpha-galactosyl (anti-Gal) is a natural human serum antibody that binds to the carbohydrate Gal alpha 1,3Gal beta 1,4GlcNAc-R (alpha-galactosyl epitope) and is synthesized by 1% of circulating B lymphocytes in response to immune stimulation by enteric bacteria. We were able to purify secretory anti-Gal from human colostrum and bile by affinity chromatography on silica-linked Gal alpha 1,3Gal beta 1,4GlcNAc. We found similar secretory anti-Gal antibodies in human milk, saliva, and vaginal washings. Secretory anti-Gal from milk and saliva was exclusively immunoglobulin A (IgA); that from colostrum and bile also contained IgG and IgM isotypes. Serum was also found to contain anti-Gal IgM and IgA in addition to the previously reported IgG. Anti-Gal IgA purified from colostrum and bile had both IgA1 and IgA2. Secretory anti-Gal from saliva, milk, colostrum, and bile agglutinated rabbit erythrocytes (RRBC) and bound to bovine thyroglobulin, both of which have abundant alpha-galactosyl epitopes. The RRBC-hemagglutinating capacity of human saliva, milk, bile, and serum was specifically adsorbed by immobilized Gal alpha 1,3Gal beta 1,4GlcNAc but not by Gal alpha 1,4Gal beta 1,4GlcNAc, Gal beta 1,3GalNAc, Gal beta 1,4GlcNAc, Gal beta 1,4GlcNAc alpha 1,2Man, or Fuc alpha 1,2Gal beta 1,4GlcNAc. No RRBC-hemagglutinating activity could be detected in rat milk, rat bile, cow milk, or rabbit bile, suggesting a restricted species distribution for secretory anti-Gal similar to that found for serum anti-Gal. Colostral anti-GaI IgA bound strongly to a sample of gram-negative bacteria isolated from the throats and stools of well children as well as to an Escherichia coli K-1 blood isolate. Colostral anti-GaI IgA inhibited the binding of a Neisseria meningitidis strain to human buccal epithelial cells, suggesting that this antibody may play a protective role at the mucosal surface.
Novel carbohydrate antigen expressions were observed on T lymphocytes from HIV infected patients using flowcytometric analysis with four mAbs; BM-1, ACFH-18, FH-2 and C-6. These carbohydrate antigens were also expressed on oncogenic transformed cells but were either not expressed or were weakly expressed in lymphocyte populations from healthy subjects. A dramatic change in glycosylation was induced on CD8+T cells from HIV infected patients. The glycosylation change correlated with the progression of the disease. The incidence of Ley antigen expression on CD8+T cells increased as the disease progressed with the ongoing impairment of immune function. The phenotype change that occurred with Ley antigen expression might reflect the abnormal activation of T lymphocytes of some specific, but unknown, population of CD8+T cells. Thus, carbohydrate changes on the cell surface may induce immunological abnormality and accelerate the damage within the CD4+T cell subset, resulting in an impairment of the antigen specific immune system.
The glycosylation pattern of the external envelope glycoprotein of human immunodeficiency virus type 2 (HIV-2) was studied in dependence on host cells and virus isolates. Strains HIV-2ALT, HIV-2ROD and HIV-2D194, differing in their biological properties and in the amino acid sequences of their env genes, were propagated in MOLT4, HUT78 and U937 cells, in human peripheral blood lymphocytes and monocytes/macrophages in the presence of [6-3H]glucosamine. Radiolabelled viral glycoproteins were isolated from the cell-free supernatants and digested with trypsin. Glycans were sequentially liberated by endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F, and fractionated according to charge and size. Comparison of the oligosaccharide profiles revealed that the envelope glycoproteins of different virus isolates, propagated in the same host cells, yielded very similar glycan patterns, whereas cultivation of an isolate in different host cells resulted in markedly divergent oligosaccharide maps. Variations concerned the proportion of high-mannose-, hybrid- and complex-type substituents, as well as the state of charge and structural parameters of the complex-type species. As a characteristic feature, complex-type glycans of macrophage-derived viral glycoprotein were almost exclusively substituted by lactosamine repeats. Hence, glycosylation of the HIV-2 external envelope glycoprotein seems to be primarily governed by host cell-specific factors rather than by the amino acid sequence of the corresponding polypeptide backbone.
Glycosylation is necessary for HIV-1 gp120 to attain a functional conformation, and individual N-linked glycans of gp120 are important, but not essential, for replication of HIV-1 in cell culture. We have constructed a mutant HIV-1 infectious clone lacking a signal for N-linked glycosylation in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by ConA and a conformation dependent human antibody IAM-2G12. This suggests that the N-linked glycan in the V1-loop modulates the three-dimensional conformation of gp120, without changing the overall functional integrity of the molecule.
There is a large body of suggestions that complex carbohydrates play a role in the regulation of cell adhesion and cell proliferation. Many reports have emphasized that proteoglycans, glycoproteins or glycolipids are participating to cell adhesion mechanisms. The use of polyvalent anti-carbohydrate antibodies and plant lectins as well as the use of glycosylation inhibitors suggested that cell proliferation can be modulated by surface carbohydrates. The dating experiment of Burger and Noonan (1970) showing restoration of contact inhibition of malignant cells by monovalent concanavalin A was a determining experiment. However, in the latter as in the others, no precise mechanism was demonstrated how carbohydrates can be involved in adhesion and proliferation. New insights were opened with the discovery of vertebrate membrane-bound and soluble lectins. The latter generally display agglutinating activities in in vitro systems, suggesting that they were potential cell adhesion molecules, by forming bridges between cell surface carbohydrates. These polyvalent molecules may be also considered as clustering agents for their cell surface ligands, consequently generating signals for cell proliferation and/or differentiation.
In past studies we demonstrated that monoclonal antibody 6F9 is a surrogate image of the meningococcal C capsular polysaccharide. These studies indicated that immunization with this anti-id resulted in a T-dependent antibody response. In the studies reported in this paper, we show that the response which is elicited is protective. Using a model of meningococcal infection in BALB/c mice in which the animals are rendered susceptible with iron dextran, we studied the ability of this anti-id to protect adult mice against challenge. These studies encompassed the ability of 6F9 to prime neonatal mice and provide them with protection to later challenge. Adult BALB/c mice immunized with 6F9 had a 100% survival and a significantly reduced level of bacteremia at 24 hours. Neonatal mice primed within 24 hours of birth and immunized at 4 weeks of age with 6F9 had a 100% survival and cleared their bacteremia by 8 hours. Neonatal mice primed with 6F9 and challenged at 5 weeks had a 90% survival. These data indicate that anti-id 6F9 is a surrogate antigen for the meningococcal C polysaccharide and is capable of inducing protective immunity in immunologically mature as well as immature animals.
Carbohydrate side chains of envelope glycoproteins of HIV-1 and other viruses have been postulated to interfere with binding of neutralizing antibodies. So far, however, little evidence for interference of specific N-glycans with virus neutralization has been provided. We used four infectious HIV-1 molecular clones chimeric for their gp 120 V3 domains to study the influence on HIV-1 neutralization of an N-glycan localized within the V3 loop. Two clones lacking the 301N-glycan were at least 8-fold more sensitive to neutralization by two V3-specific monoclonal antibodies (MAbs) and 2- to 10-fold more sensitive to neutralization by a CD4-binding-site-specific human MAb than two HIV-1 clones glycosylated at this site. The affinity of the V3 MAbs for soluble gp120 of the four clones was similar. However, a decreased binding of these MAbs to the gp120 of the two 301N-glycosylated clones was observed when the majority of gp120 was virion-associated during the initial binding step. These findings indicate that the 301N-glycan may interfere with the binding of neutralizing antibodies by limiting the accessibility of neutralization sites or by inducing conformational changes in the HIV-1 gp120 molecule.
The gp160 envelope glycoprotein of human immunodeficiency virus type-1 (HIV-1) is an essential component of current vaccine trials. The glycans of gp160, part of which are highly sialylated, have been shown to influence gp160 immunogenicity. Here, using a panel of synthetic V3 peptides, we characterized the anti-V3 antibodies generated in rabbits immunized by desialylated recombinant gp160LAI. Amino acid residues flanking the GPGR tip of V3 were necessary for the recognition by anti-V3 antibodies raised against either the native or desialylated gp160. Both types of antibodies reacted to V3 peptides of MN and SF2 strains and with a North American/European V3 consensus peptide, while anti-desialylated gp160LAI antibodies reacted in addition to the V3 of CDC4, WMJ2 and NY5 strains. Yet, the V3 peptides did not significantly differ in their secondary structure, as determined by circular dichroism. The titer and avidity for V3MN of anti-desialylated gp160LAI antibodies were significantly lower than those of anti-native gp160LAI, which likely accounts for the inability of anti-desialylated gp160LAI sera to neutralize HIV-1MN-induced syncytia. These results indicate that V3 immunogenicity may be influenced by subtle directed changes in the gp160 glycosylation pattern.
The unusually highly glycosylated state of the major envelope glycoprotein (gp160) of the human immunodeficiency virus has offered a challenge to both glycobiologists and virologists. What is the functional significance of such a mass of glycans and how might they be manipulated to disadvantage virus pathogenesis? Some answers to each of these questions have already been obtained: N-linked glycans are necessary for the creation, but not the maintenance, of a bioactive conformation, and drug-induced alteration of the glycosylation pattern can lead to impaired virus infectivity. As a model for studying glycan function and as a target for antiviral therapy, gp160 represents a unique candidate.
The concept of a common mucosal immune system, through which specific antigen-activated lymphocytes from the gut can disseminate immunity both along the intestinal tract and to various other mucosal and glandular tissues, has generated much current interest in the possibility of developing oral vaccines, not only for enteric infections but also for infections in the respiratory and urogenital tracts. However, to date it has proven difficult in practice to stimulate strong mucosal IgA immune responses by either parenteral or oral-mucosal administration of most antigens, and experience with soluble protein antigens has, on the whole, been disappointing. A notable exception in this regard is cholera toxin (CT) and in humans more than in other species, its nontoxic B subunit pentamer moiety (CTB). Based on this, CTB has become an important component in recently developed oral vaccines against cholera as well as against diarrhea caused by enterotoxigenic Escherichia coli producing CT-like heat-labile enterotoxin(s). Since the strong immunogenicity of CT and CTB can, to a large extent, be explained by their ability to bind to receptors on the intestinal mucosal surface, there has recently been much interest in approaches using CTB as an oral delivery carrier system for other vaccine-relevant antigens, and much progress has been made in preparing immunogenic hybrid proteins by coupling various protein or peptide antigens chemically or genetically to CTB. Indeed, in several systems, oral administration of such hybrid antigens has been found to markedly potentiate both intestinal and extraintestinal IgA immune responses against the CTB-coupled antigens and also to elicit substantial circulating antibody responses. Besides the mucosal immunopotentiating effect of either CT or CTB owing to their similar capacity as oral antigen-delivery vehicles, CT, but in most systems tested not CTB, also has strong adjuvant properties for stimulating mucosal IgA immune responses to admixed (not coupled) unrelated antigens after oral immunization. This adjuvant activity appears to be closely linked to the ADP-ribosylating action of CT (and specifically of its A subunit) leading to enhanced cyclic AMP formation in the affected cell, and efforts to eliminate the enterotoxic activity without losing adjuvanticity have so far not met with success.
Our recent studies have indicated that galactosyl ceramide (GalCer) or sulfatide (sul) may serve as an alternate receptor for human immunodeficiency virus (HIV) in neural cells. In this paper, we describe the mapping of GalCer/sul binding region of HIV env glycoprotein gp120. Deglycosylated gp120 binds to GalCer, suggesting that the amino acids of glycoprotein gp120 and not the carbohydrates are responsible for the observed binding. Specific regions of gp120 responsible for the binding were analyzed by using varying-length truncations of gp120 expressed in Escherichia coli and vaccinia virus. Purified recombinant gp120 containing amino acids 200-295 of gp120 bind to GalCer/sul, whereas recombinant env proteins that deleted this region did not bind. These recombinant proteins also bind to SK-N-MC-derived neuroblastoma cells, the binding of which is inhibited by anti-GalCer. In addition, 125I-labeled gp120 binding to GalCer is inhibited by these proteins. Studies using lysates containing truncated gp120 expressed in vaccinia virus also gave similar results. By eliminating the overlapping regions that do not bind, we conclude that the amino acids responsible for GalCer/sul binding reside between amino acids 206 and 275. The significance of this mapping is discussed in relation to the neurotropism of HIV.