Glycosylation and biological activity of CAMPATH-1H expressed in different cell lines and grown under different culture conditions.

CAMPATH-1H (where CAMPATH is a trade mark of Wellcome group companies), a humanized IgG antibody used in the therapy of lymphoma, leukaemia and rheumatoid arthritis, has been expressed in Chinese hamster ovary, Y0 myeloma and NS0 myeloma cell lines. These engineered cell lines were grown under different culture conditions, and the antibody isolated and purified. N-Linked oligosaccharides, on the CH2 heavy chain region of the antibody, were isolated and analysed by hydrazinolysis, high-performance anion-exchange chromatography with pulsed amperometric detection, laser-desorption mass spectrometry and sequential exoglycosidase treatment. Both the glycosylation pattern and the biological activity of CAMPATH-1H, as measured by antibody-dependent cell-mediated cytotoxicity, were markedly affected by the cell line used to express the antibody. It is concluded that glycosylation of the antibody may be important in the clinical outcome of therapy.

Primary structure of CD52.

The CD52 antigen was extracted from human spleens with organic solvents and purified by immunoaffinity and reverse-phase chromatography. The latter step resolved two CD52 species, called CD52-I and CD52-II. Both species were found to contain similar N-linked oligosaccharides and glycosylphosphatidylinositol (GPI) anchor glycans. The N-linked oligosaccharides were characterized by methylation linkage analysis and, following exhaustive neuraminidase and endo-beta-galactosidase digestion, by the reagent array analysis method. The results showed that the single CD52 N-glycosylation site is occupied by large sialylated, polylactosamine-containing, core-fucosylated tetraantennary oligosaccharides. The locations of the phosphoryl substituents on the GPI anchor glycan were determined using a new and sensitive method based upon partial acid hydrolysis of the GPI glycan. The difference between CD52-I and CD52-II was in the phosphatidylinositol (PI) moieties of the GPI anchors. The phosphatidylinositol-specific phospholipase C-sensitive CD52-I contained exclusively distearoyl-PI, while the PI-phospholipase C-resistant CD52-II contained predominantly a palmitoylated stearoyl-arachidonoyl-PI, as judged by electrospray ionization mass spectrometry. Tandem mass spectrometric studies indicated that the palmitoyl residue of the CD52-II anchor is attached to the 2-position of the myo-inositol ring. Both the CD52-I and CD52-II PI structures are unusual for GPI anchors and the possible significance of this is discussed. The alkali-lability of the CD52 epitope recognized by the Campath-1H monoclonal antibody was studied. The data suggest that the alkali-labile hydroxyester-linked fatty acids of the GPI anchor are necessary for antibody binding.

Structure of the CAMPATH-1 antigen, a glycosylphosphatidylinositol-anchored glycoprotein which is an exceptionally good target for complement lysis.

CAMPATH-1 antibodies recognize a unique molecule on human lymphocytes and are unusually efficient at causing cell lysis with homologous complement. They have been successfully used for lymphocyte depletion in vivo in a variety of diseases. We find that the antigen is a very small glycosylphosphatidylinositol (GPI)-anchored glycoprotein with a mature peptide comprising only 12 amino acids. It can be separated into two distinct antigenic fractions which differ in their susceptibility to phosphatidylinositol-specific phospholipase C. There is one N-linked glycosylation site, but no evidence for O-glycosylation despite the presence of several serine and threonine residues. The antibodies were found to bind, albeit with a generally reduced affinity, to a proteolytic fragment containing the C-terminal tripeptide and the GPI anchor. We postulate that one of the reasons why the CAMPATH-1 antibodies are so good for cell lysis is because they bind to an epitope which is likely to be very close to the lipid bilayer.

Galactose oxidation in the design of immunogenic vaccines.

Potent immunological adjuvants are urgently required to complement recombinant and synthetic vaccines. However, it has not been possible to derive new principles for the design of vaccine adjuvants from knowledge of the mechanism of immunogenicity. Carbonyl-amino condensations, which are essential to the inductive interaction between antigen-presenting cells and T helper cells, were tested as a target for the enhancement of immune responses. Enzymic oxidation of cell-surface galactose to increase aminereactive carbonyl groups on murine lymphocytes and antigen-presenting cells provided a potent, noninflammatory method of enhancing the immunogenicity of viral, bacterial, and protozoal subunit vaccines in mice.

Inhibition of specific T-cell activation by monosaccharides is through their reactivity as aldehydes.

The effect of monosaccharides on the inductive interaction between antigen-presenting cells and T cells was investigated in a human system. Some monosaccharides, but not others, were found to inhibit antigen-specific T-cell activation. Responses to mitogen were unaffected. In order for inhibition to occur, a high concentration (approximately 50 mM) of monosaccharide was necessary. The role of monosaccharide aldehyde groups in inhibition was investigated using the alpha-methyl pyranoside and the alditol forms of inhibitory monosaccharides. Unlike the native monosaccharides, these molecular configurations possess the ring structure and the open chain structure respectively but do not contain aldehydes. Together they represent all the molecular characteristics of both forms of the monosaccharide except the possession of aldehyde groups. These two molecular species produced no significant inhibition. Modified forms of the sugar moiety of ribofuranosidoadenine (adenosine) were also tested. The periodate oxidized form of the molecule in which the ribose bears two aldehyde groups, was a potent inhibitor of antigen-induced T-cell activation whereas periodate-oxidized, borohydride-reduced ribofuranosidoadenine, in which the ribose aldehydes are converted to alcohols, produced no inhibition. The former was shown to form Schiff bases with ligands on peripheral blood mononuclear cells (PBMC) as predicted whereas the latter did not. Periodate oxidized dextran, but not native dextran, was also inhibitory. Together these data show that inhibition of T-cell activation by sugars requires reactive aldehydes and this is consistent with the Schiff base model of specific antigen-presenting cell (APC)-T cell inductive interaction in which exogenous aldehydes and other carbonyl donors prevent the necessary formation of Schiff bases between cellular ligands.

Murine cross-reactive T-cell epitopes of Neisseria meningitidis outer membrane proteins.

Five non-covalent vaccines of outer membrane proteins (OMPs) complexed to capsular polysaccharide were prepared from Neisseria meningitidis serogroup B strains. Each vaccine contained distinct serotype (class 2/3) and subtype (class 1) OMPs. The cross-reactivity of the T-cell response to the meningococcal vaccine-associated proteins was examined in an in vitro T-cell proliferative assay, following antigenic priming of mice with one of these vaccines (MB6:P1.6) or with its purified class 1 (subtype P1.6) and class 2 (serotype 6) proteins. Cross-reactive T-cell epitopes were found in all five vaccine preparations on both the class 1 and class 2/3 OMPs. Priming of mice with the subtype P1.6 N-terminal peptide led to a significant but small increase in T-cell proliferation with the MB6:P1.6 vaccine.

Specificity of the immune response to the group B polysaccharide of Neisseria meningitidis.

A panel of monoclonal antibodies (mAb) and polyclonal sera of murine, human and equine origin, of IgM isotype and with specificity for Neisseria meningitidis group B polysaccharide, an alpha(2----8)-linked homopolymer of sialic acid, were examined for their antigenic and biological specificities. The nature of the antigenic determinants on B polysaccharide was investigated using a series of N-acyl derivatives of B polysaccharide, two sialic acid polymers containing alpha(2----9)-linkages and a series of polynucleotides. The panel of antibodies recognized an array of unrelated antigenic determinants on the B polysaccharide, despite its structural simplicity, and all but one were highly effective in an in vitro bactericidal assay and/or in an in vivo murine passive protection model. There was no evidence that B polysaccharide induced antibody capable of blocking biological activity (blocking antibody).

Immunogenicity in adult males of a Neisseria meningitidis group B vaccine composed of polysaccharide complexed with outer membrane proteins.

Twenty five adult male volunteers were given a vaccine composed of the capsular B polysaccharide non-covalently complexed to serotype 6 outer membrane proteins (OMP) of Neisseria meningitidis. Subjects were divided into three dose groups receiving 50, 100 or 150 micrograms vaccine in aluminium hydroxide in each of three injections spaced 4 weeks apart. Systemic signs/symptoms considered clinically significant were recorded on 6% (4/70) of occasions and were succeeded by withdrawal of two volunteers from the study. Local injection site reactions, mostly mild to moderate, were reported after all vaccinations with one such reaction leading to a third volunteer withdrawing from the study. Geometric mean anti-B responses before immunization and 1 week after the third immunization (9 weeks) were 3.60 and 7.12 micrograms ml-1 in the 50 micrograms group (p less than 0.05) 2.05 and 12.19 micrograms ml-1 in the 100 micrograms group (p less than 0.001), and 3.68 and 14.20 micrograms ml-1 in the 150 micrograms group (p less than 0.001). The anti-B response was predominantly of the IgM isotype and persistence above prevaccination levels was evident for at least 12 months. Anti-type 6 OMP responses were also evidenced with geometric mean multiplicative increases over prevaccination levels at 9 weeks and 6 months of 7.8 and 4.2 for the 50 micrograms group, 11.6 and 5.6 for the 100 micrograms group and 6.8 and 3.4 for the 150 micrograms group. The bulk of this response was of the IgG isotype. Passive protection of mice was achieved with both pre- and post-vaccination (9 weeks; 100 and 150 micrograms groups) pools of sera.(ABSTRACT TRUNCATED AT 250 WORDS)

Passive transfer of meningococcal group B polysaccharide antibodies to the offspring of pregnant rabbits and their protective role against infection with Escherichia coli K1.

Pregnant rabbits vaccinated with meningococcal group B polysaccharide complexed to outer membrane proteins (serotype 6) responded to produce IgG, IgM and IgA anti-B polysaccharide antibodies, which were passively transferred to the offspring (IgG preferentially) and could be detected in their sera immediately after birth. These antibody levels were sustained in the mothers but diminished in the offspring to background levels at day 22 after birth. In a subsequent experiment, rabbits immunized with the group B vaccine had offspring that proved considerably more resistant to infection with Escherichia coli K1 than the control litters from non-immune mothers. Although not complete, protection was statistically of high significance and correlated well with the anti-B polysaccharide titres obtained in the mothers.

Immune responses in mice to different noncovalent complexes of meningococcal B polysaccharide and outer membrane proteins.

Noncovalent complexes of meningococcal group B polysaccharide and outer membrane proteins (OMPs) from serotypes 2a, 2b, 6, 8, and 15 were made either by coextraction of the two components from culture supernatants or by separate preparation of both OMPs and B polysaccharide with subsequent mixing of the two components. The former method showed a markedly greater degree of binding of B polysaccharide to OMPs, a less heterogeneous composition of OMPs, and a lower lipopolysaccharide content. Immunization of mice with these complexes showed that a higher degree of binding of B polysaccharide to OMPs resulted in a higher anti-B response. Western blotting (immunoblotting) and solid-phase radioimmunoassay techniques confirmed that antibody cross-reactions occur among OMP serotypes. The occurrence of common T-cell determinants between OMP serotypes was suggested when mice primed with a type 6 OMP complex and challenged with a homologous or heterologous serotype complex responded with significantly higher anti-B titers than unprimed animals. These memory T cells persisted for at least 12 months in mice.

Murine monoclonal antibodies for detection of antigens and culture identification of Neisseria meningitidis group B and Escherichia coli K-1.

Four murine monoclonal antibodies which reacted with a (2----8)alpha-linked sialic acid polysaccharide were produced. Three of the antibodies reacted specifically with Neisseria meningitidis serogroup B and Escherichia coli K-1 polysaccharide antigens, whereas one antibody cross-reacted with N. meningitidis group C polysaccharide antigen, a (2----9)alpha-linked homopolymer of sialic acid. By using the most avid antibody (MB 62), a latex particle agglutination test was developed which could detect capsular polysaccharide at 10 ng/ml. It also detected antigen in the cerebrospinal fluid (CSF) of all seven N. meningitidis group B- and two E. coli K-1-infected patients, whereas 57 control CSF samples, including 8 from neonates, were negative. Cultures of 21 N. meningitidis group B strains, 7 E. coli K-1 strains, and 1 Moraxella nonliquefaciens strain gave a positive result, whereas 53 strains from other serogroups were all negative. In a separate clinical evaluation, the overall sensitivity of the latex particle agglutination test was 81% (22 of 27) with fresh CSF samples, 48% (15 of 31) with stored CSF samples, and 94% (32 of 34) with blood cultures. No false-positive reactions were recorded with 165 control CSF samples, and the specificity with blood cultures was 99.4% (519 of 522).

An integrated molecular and immunological approach towards a meningococcal group B vaccine.

There has been a notable lack of success in producing an effective vaccine against Neisseria meningitidis group B infections, despite such prophylaxis being available for group A and C disease. The reasons for this are reviewed and evidence presented that a vaccine based on the group B capsular polysaccharide should be pursued. To be effective, a clear understanding of, and improvement in the poor immunogenicity of the polysaccharide is required. Consequently, the nature of the antigenic structure involved in immune recognition has been evaluated at the molecular level and reasons for the poor immunogenicity of the B polysaccharide are presented. Methods of increasing the immunogenicity are proposed with the intention of undertaking human volunteer trials.

Analysis of the chain length of oligomers and polymers of sialic acid isolated from Neisseria meningitidis group B and C and Escherichia coli K1 and K92.

A series of (2----8)-alpha-, (2----9)-alpha-, and alternate (2----8)-alpha- and (2----9)-alpha-linked oligomers of sialic acid (N-acetylneuraminic acid, NeuNAc) was prepared by digestion with bacteriophage or by partial hydrolysis at pH 7.0 and 100 degrees of polymers of sialic acid produced by Neisseria meningitidis and Escherichia coli. The oligosaccharides were purified by gel filtration or by anion-exchange chromatography, and their chain lengths were determined by colorimetric measurement of the formaldehyde released from the non-reducing end residue after periodate oxidation, radiolabelling of the reducing end residue by reduction with borotritiide, and determination of the ratio of the non-reducing end and internal residues by g.l.c. of the trimethylsilyl derivatives of the methyl ester methyl beta-ketosides. 1H-N.m.r. spectroscopy was used to confirm the chain length of two oligosaccharides. These methods were used to determine the average chain-length of the sialic acid polysaccharides produced by N. meningitidis and E. coli and the percentage of chains with covalently bound lipid moieties at the reducing end.

Thymic-dependence and immune memory in mice vaccinated with meningococcal polysaccharide group B complexed to outer membrane protein.

Athymic mice were capable of producing IgM antibodies to B polysaccharide of N. meningitidis when immunized with the polysaccharide complexed to outer membrane proteins, whereas no immunogenicity could be demonstrated with the purified polysaccharide. Transfer of T cells to athymic cells demonstrated that the primary anti-B response was truly thymus-independent but the bulk of the secondary response was thymus-dependent. Transfer of T and B cells purified from spleens of immune mice into irradiated recipients showed the presence of memory T cells, but no memory B cells could be found.

Structural and conformational features of the Escherichia coli K92 capsular polysaccharide.

Escherichia coli K92 polysaccharide, an alternating (2----8)-alpha- and (2----9)-alpha-linked heteropolymer of sialic acid, undergoes lactonisation (10-20%) at low pH or on treatment with a water-soluble carbodi-imide (greater than 90%). Under the latter conditions, the formation of O-acylisourea is a minor pathway. The antigenicity of the K92 polysaccharide is unaffected by lactonisation of approximately 10% of the residues, but is abolished by lactonisation of approximately 90% of the residues. The unexpected, relative resistance of the K92 polysaccharide to periodate oxidation contrasts with the behaviour of the meningococcal non-O-acetylated (O-Ac-) C polysaccharide, a (2----9)-alpha-linked homopolymer of sialic acid. Two-dimensional 13C-1H-n.m.r. correlation spectroscopy has been used to assign 1H chemical shifts which have aided the interpretation of a resolution-enhanced one-dimensional spectrum. This has led to a determination of conformational features of the K92 polysaccharide in solution. The side-chain adopts a conformation such that H-7 and H-8 are gauche in the (2----8)-alpha-linked residues (1a or 1b), but antiperiplanar in the (2----9)-alpha-linked residues (2). Molecular correlation times have been calculated and some aspects of internal motion elucidated.

Effect of aluminum ions on chemical and immunological properties of meningococcal group B polysaccharide.

Soluble salts of aluminum were examined for their capacity to complex with purified meningococcal group B polysaccharide. The formation of the complexes resulted, first, in a markedly reduced rate of internal esterification at acid pH and, consequently, prolonged stability of the antigen as measured by its reactivity with antibody at pH 4 and, second, in an increased resistance to neuraminidase. Al3+ complexes of B polysaccharides were tested for immunogenicity in mice and found to be no better than the purified polysaccharide in the Na+ or Ca2+ form. However, when Neisseria meningitidis type 6 protein (outer membrane) complexed to B polysaccharide was tested, a substantial increase in anti-B titers was detected, whereas antiprotein titers remained unchanged. The possibility of using combinations of metal-polysaccharide-outer membrane protein complexes as vaccines for humans is discussed.

Immunity and protection of mice against Neisseria meningitidis group B by vaccination, using polysaccharide complexed with outer membrane proteins: a comparison with purified B polysaccharide.

A methodology for preparing outer membrane proteins (type specific) complexed to group B polysaccharide of Neisseria meningitidis is described. These complexes, low in nucleic acid and lipopolysaccharide content, were immunogenic in mice with induction of humoral antigroup B and antiprotein responses. Immunized mice were also protected against challenge with N. meningitidis group B strains of the same or a different type from that used for vaccination. Both immunity and protection were enhanced when the mice received a secondary immunization with the protein-polysaccharide complex. Additional data have shown the capacity of purified B polysaccharide to induce immunological memory, even though it is incapable of inducing a humoral response when given alone.

Rate, mechanism, and immunochemical studies of lactonisation in serogroup B and C polysaccharides of Neisseria meningitidis.

Meningococcal Serogroup B polysaccharide and colominic acid, which are (2----8)-alpha-linked homopolymers of sialic acid, undergo lactonisation at low pH at a rate which is dependent upon the molecular size and upon the salt form (Na+ or Ca2+). Meningococcal Serogroup C polysaccharide, a (2----9)-alpha-linked homopolymer of sialic acid with acetyl groups present at O-7 and/or O-8, reacts with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to give an O-acylisourea. The rate of formation of O-acylisourea does not differ substantially between O-acetylated (O-Ac+) C, non-O-acetylated (O-Ac-) C, and B polysaccharide. 13C-N.m.r. spectroscopy shows that, in the absence of O-acetyl groups, the majority of the activated carboxyl groups of C polysaccharide condense with an adjacent HO-8 to form a delta-lactone. Immunochemical studies show that the antigenicity of B polysaccharide is markedly reduced on lactonisation of less than 20%, as measured by a radioimmunoassay using an anti-B monoclonal antibody, and that low-molecular-weight colominic acid is poorly antigenic both before and after lactonisation, suggesting the presence of conformational determinants on B polysaccharide. In contrast, lactonisation and/or formation of O-acylisourea groups in the (O-Ac+)-C polysaccharide does not cause a significant decrease in the antigenicity, which is consistent with a sequential (structural) determinant on the molecule.

Conformational and dynamic differences between N. meningitidis serogroup B and C polysaccharides, using n.m.r. spectroscopy and molecular mechanics calculations.

1H-N.m.r. spectroscopy has been used to determine the conformation in aqueous solution of the sialic acid residues of the N. meningitidis serogroup B and non-O-acetylated (O-Ac-)-C polysaccharides, and of N-acetylneuraminic acid (NeuNAc). In all cases, the sugar adopts the 2C5 conformation. The side-chain of NeuNAc adopts a conformation such that H-7 and H-8 are approximately anti-periplanar. This conformation is also found in the (O-Ac-)-C polysaccharide, whereas H-7 and H-8 are gauche in the B polysaccharide. Molecular mechanics calculations have been used to probe the conformational preferences of the variously linked sialic acid residues, and the results are in general agreement with those based on the 1H-n.m.r. data. The 13C-n.m.r. spin-lattice relaxation-times have been interpreted in terms of the molecular dynamics of the B and (O-Ac-)-C polysaccharides. Molecular correlation times have been calculated and details of internal rotational or segmental motion elucidated. The C polysaccharide is characterised by internal or segmental motion in the C-7 to C-9 side-chain of the sialic acid repeating-unit, whereas the B polysaccharide has little or no such movement and tumbles in solution as a rigid species with internal rotation of only the pendant C-9 group. The conformational differences suggest a substantially different three-dimensional structure in solution for these polysaccharides.