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T E Michaelsen

Publications and source records attributed to T E Michaelsen.

At least 55 records · Page 3Linked to original sources
PubMed1997

Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells.

We have developed new cassette expression vectors for the cloning of any intact V-region gene followed by any C-region gene. Both the heavy-and light chain vectors harbor a strong hCMV promoter, restriction site cassettes for cloning of both V- and C-region genes, transcription termination signals, fl-ori for single stranded DNA (ssDNA) synthesis, selection marker for Neomycin and SV40 ori for transient expression. The vectors accept VH and VL chain genes obtained by RT-PCR. Reamplification of the V genes is then performed with a new set of primers which are designed specifically for each individual V gene. Cloning into the vectors is aided by restriction sites located just outside the V-gene coding region, thus keeping the V-genes intact. The vectors also contain cloning sites for the exchange of genomic C-genes so that the resulting Ig genes may code for complete antibodies, antibody fragments or fusion proteins. A simple subcloning step permits the expression of both heavy and light chain genes from one single vector, thus avoiding co-transfection of the two vectors. The usefulness of the vectors was confirmed by construction of mouse-human chimeric antibodies. The V-genes were derived from a hybridoma cell line, TP-3, and was combined with human C kappa, C gamma 3 and C gamma 1 genes as well as with CH1 gamma 3. High yields of recombinant antibody products in NSO cells were obtained. Transient expression was also demonstrated.

Animals↗
PubMed1997

CDw78--a determinant on a major histocompatibility complex class II subpopulation that can be induced to associate with the cytoskeleton.

In the present study we demonstrate that CDw78 monoclonal antibody (mAb) recognizes a distinct subpopulation of major histocompatibility complex (MHC) class II molecules. We show that the CDw78 epitope is present on less than 10% of the total number of MHC class II molecules expressed on different cells, is not linked to a single isotype, and exhibits a characteristic expression pattern in tonsils. While mAb against MHC class II (DR, DP and DQ) stained the majority of cells both in the mantle zone and in germinal centers, the CDw78 staining was more heterogeneous with the strongest reactivity and the highest number of positive cells in the mantle zone and in the light centrocyte-rich part of the germinal centers. Antibodies to this MHC class II subpopulation (e.g. FN1) induced association with the cytoskeleton and a subsequent capping in more than 90% of peripheral blood B cells. In contrast, mAb against MHC class II (DR, DP and DQ) did not induce association with the cytoskeleton and only 10-20% of B cells were induced to cap, suggesting that CDw78 defines a population of MHC class II molecules functionally different from the majority of these antigens. Scatchard plot analysis indicates that FN1 mAb is of relatively low affinity (Ka = 1.5 x 10(8) M(-1)) and monovalent Fab fragments fail to bind to the cell surface with measurable affinity. Our data seen in the context of the ability of FN1 to co-stimulate B cells with a suboptimal dose of anti-mu suggest that CDw78 mAb might recognize a functional important subpopulation of MHC class II molecules so far not described. It seems likely that this subpopulation represents dimerized or aggregated MHC class II molecules that can selectively bind this low-affinity mAb.

Antibodies, Monoclonal↗
PubMed1997

Vaccine-induced IgG antibodies to the linear epitope on the PorB outer membrane protein promote opsonophagocytosis of Neisseria meningitidis by human neutrophils.

The serotype 15 PorB protein of Neisseria meningitidis contains an N-terminal linear immunodominant B-cell epitope located on the putative loop 1 (VR1) region. This epitope has previously been shown to stimulate antibody formation in 74% of the vaccinees after three doses of the Norwegian group B outer-membrane vesicle (OMV) vaccine. In the present study, the purified PorB protein and the 23mer synthetic peptide D63b2 covering VR1 region were immobilized onto N-hydroxysuccinimide-activated matrix and used for affinity purification of the specific IgG antibodies from sera of three selected vaccinees. PorB- and peptide D63b2-specific IgG preparations bound to the PorB protein on immunoblots and reacted with strain 44/76 and OMV complexes expressing the serotype 15 PorB protein, but not with the PorB-deficient mutant, suggesting high specificity for the PorB protein. Both PorB- and peptide D63b2-specific IgG were marginally bactericidal, but enabled strong opsonophagocytosis measured as respiratory burst response of human neutrophils and internalization of opsonized FTTC-labeled meningococci. The data indicate that about 30-57% of the bulk serum opsonic activity for the 44/76 bacteria could be ascribed to linear epitope-specific IgG1, thus contributing to vaccine-induced protection against systemic meningococcal disease via the opsonophagocytic route of pathogen clearance.

Adult↗
PubMed1997

Outer membrane vesicle vaccines made from short-chain lipopolysaccharide mutants of serogroup B Neisseria meningitidis: effect of the carbohydrate chain length on the immune response.

Outer membrane vesicle (OMV) vaccines were made from Neisseria meningitidis strain 44/76 and its two short-chain lipopolysaccharide (LPS) mutants, Mu-1 and Mu-4. Only the 44/76 vaccine contained LPS with the host antigen lacto-N-neotetraose. The protein composition of the vaccines was similar. The LPS carbohydrate chain length proved to influence drastic changes in the LPS immunogenicity as well as the outer membrane proteins (OMPs) ability to elicit functional antibodies in mice. Only LPS in the Mu-1 and Mu-4 vaccines were immunogenic, and the 44/76 vaccine differed also by not inducing antibodies to the class 4 OMP. The Mu-1 vaccine, with a LPS carbohydrate chain comprising only two residues of 2-keto-3-deoxy-octonic acid, induced lower bactericidal activity and less antibodies to the class 1 OMP, compared to the two other vaccines. This indicates that LPS of a certain carbohydrate chain length is required for adequate exposure of the class 1 OMP epitopes essential for inducing bactericidal antibodies.

Animals↗
PubMed1997

Crossreactions and sequence homologies between recombinant polypeptides from Leishmania aethiopica and human IgG and IgM.

Recombinant DNA fragments M (154 bp) and G (206 bp), coding for recombinant polypeptides that crossreact with human IgM and IgG, have been isolated from a genomic library of Leishmania aethiopica. Epitope scanning of the two recombinant polypeptides, using overlapping octapeptides, revealed several crossreactive epitopes present in both recombinant proteins. By comparing amino acid sequences, similar sequences in human mu and gamma immunoglobulin heavy chains were identified. One of the parasite octapeptides is identical to an octapeptide in gamma1 covering the Gm(a) allotypic marker. Expression of both the M and G fragments was detected in the parasites by RT-PCR of total mRNA, using primers specific for these fragments. Preliminary data showed that the presence of autoimmune anti-IgG antibodies was more pronounced in sera from patients with diffuse cutaneous leishmaniasis than in sera from patients with localised cutaneous leishmaniasis. We suggest that these immunoglobulin-crossreacting epitopes potentially might contribute to the induction of rheumatoid factors and be involved in the interplay between the parasite and the host immune system.

Amino Acid Sequence↗
PubMed1996

Quantitation of IgG subclass antibody responses after immunization with a group B meningococcal outer membrane vesicle vaccine, using monoclonal mouse-human chimeric antibodies as standards.

An ELISA method was developed to quantitate gravimetrically (microgram/ml) the IgG subclass response against a Norwegian vaccine composed of outer membrane vesicles (OMV) isolated from a Neisseria meningitidis B:15:P1.7,16 epidemic strain. Chimeric mouse-human anti-hapten NIP (5-iodo-4-hydroxy-3-nitrophenacetyl) antibodies of each subclass were used for calibration purposes. Before vaccination, low amounts of IgG1 and IgG2 antibodies against OMV were detectable in all vaccinees, whereas IgG3 was only detectable in one of the 21 vaccinees. After vaccination, IgG1 antibodies dominated the response followed by IgG3 and low to moderate levels of IgG2 antibodies. IgG4 was only detectable at very low levels in a few vaccinees. All sera showed close to parallel dose-response curves to each other for IgG1 and IgG3, whereas the IgG2 curves were not parallel to chimeric IgG2 and could thus not be quantitated gravimetrically. For IgG3, 1/3 of the vaccinee sera showed non-parallel dose-response curves to the rest of the vaccinee sera and to chimeric IgG3 and could not be gravimetrically quantitated. The rest of the sera showed parallel dose-response curves with the chimeric IgG3 and gravimetric quantitation was possible. This study illustrates that chimeric antibodies can be used as calibrators to quantitate IgG subclass antibody responses against OMV in gravimetric units and that the vaccine mainly induces IgG1 and IgG3 antibodies in humans.

Adult↗
PubMed1996

Effect of the IgM and IgA secretory tailpieces on polymerization and secretion of IgM and IgG.

Pentameric IgM and dimeric IgA both contain disulfide bonds between cysteines located in the secretory tailpieces of the heavy chains. To compare the influences of the mu and alpha tailpieces on the polymeric structure, we have replaced amino acids in the tailpiece of the human mu-chain with amino acids found in the corresponding positions in the human alpha tailpiece. We show that an IgM with an alpha tailpiece (IgM L561H, Y562V, L566V, S569A, D570E, T571V, and A572D) as well as IgM L561H, Y562V, and IgM A572D have a size distribution similar to that of wild-type IgM. However, one IgM mutant with a mu/alpha hybrid tailpiece (IgM L566V, S569A, D570E, T571V, and A572D) is secreted as a mixture of mainly hexamers, pentamers, tetramers, and dimers. The tetramers and dimers are specifically formed and secreted at the expense of pentamers and hexamers; no alterations in polymerization or secretion rates were observed. We have also incorporated the mu, alpha, and hybrid mu/alpha tailpieces to a human IgG3 or IgGL309C mutant. The IgG-tailpiece mutants are poorly secreted, but the secreted fractions contain multimeric molecules. Each of the mutants that contain both the L309C mutation and a secretory tailpiece forms mainly hexamers; however, small differences in polymer distribution exist for the different tailpieces. Comparison of the influence of different tailpieces on IgM and IgG polymeric structures suggests that the function of a specific tailpiece is dependent on other parts of the heavy chain, which can vary for different isotypes.

Amino Acid Sequence↗
PubMed1996

Comparison of surface properties of human IgA, IgE, IgG and IgM antibodies with identical and different specificities.

In this paper, the authors report the use of liquid-liquid partition chromatography (LLPC) in an aqueous polyethylene glycol (PEG)/dextran two-phase system to compare the surface properties (partition properties) of human antibodies and fragments thereof. The surface properties of all the monoclonal antibodies of different classes and subclasses investigated were within the same broad range as that observed for the polyclonal antibodies and no relationship was found between the exposed surfaces of the immunoglobulins (Ig) and their heavy chain isotype. Moreover, Fc fragments from various IgG1, 2 and 4 myeloma proteins were found to exhibit similar surface properties. Employing chimeric antibodies with identical variable regions the authors found that intact IgG1, 2 and 4 displayed identical surface properties, while the corresponding IgA1, IgA2, IgG3, IgE and IgM antibodies differed both from each other and from the IgGs. The surface properties of chimeric IgG3 could be made similar to those of the IgG1, 2 and 4 chimers by partially reducing the length of the hinge section, but new differences in surface properties appeared when their hinges were of similar length. Thus, LLPC can be used to detect differences or similarities in the surface properties of the antigen-binding regions as well as the Fc part in the various isotypes. This can shed light on biological activities such as antigen binding and effector function.

Antibody Specificity↗
PubMed1996

Immune responses to linear epitopes on the PorB protein of Neisseria meningitidis in patients with systemic meningococcal disease.

Neisserial porins, the major protein constituents of the outer membrane capable of inducing antibody responses in humans, are considered to be meningococcal vaccine candidates, so it is important to map the relevant B-cell epitopes. For B-cell epitope analyses of the serotype 15 PorB protein in Neisseria meningitidis, paired sera from selected patients with systemic meningococcal disease (SMD) were screened with synthetic 12mer peptides spanning the PorB protein sequence, and/or its variable region 1 (VR1). A 'SMD-related' linear B-cell epitope was found within the VR1 region consisting of 14 residues (17svFHQNGQVTEvtt30). A 23mer soluble peptide (D63b2) that covered the VR1 region, including the complete 17svFHQNGQVTEvtt30 sequence, was recognized, whereas no detectable binding was observed to a 16mer peptide (D63a1) containing most of the essential sequence (19FHQNGQVTEvtt30). A low frequency of IgG responses specific for the PorB linear epitopes was found in convalescent-phase sera from 132 SMD patients studied, as judged from both immunoblotting studies (24/132; 18.2%) and reactivity with peptide D63b2 (18/132; 13.6%). Peptide D63b2 significantly inhibited IgG binding to the denatured PorB protein on immunoblots, suggesting that this B-cell epitope was one of the main linear epitopes on the PorB protein recognized by sera from some SMD patients.

Adolescent↗
PubMed1995

The structural requirements for complement activation by IgG: does it hinge on the hinge?

The flexibility of antibody molecules principally derives from the structure of the hinge region. It has generally been accepted that the flexibility of the IgG hinge is necessary for complement activation to occur; however, recent studies dispute this premise. As described here by Ole Henrik Brekke, Terje Michaelsen and Inger Sandlie, it now appears that the only requirement of the hinge region for complement activation is the presence of inter-heavy-chain disulfide bond(s). Furthermore, the structural basis for the differences between IgG subclasses with respect to effector functions appear to be located within the CH2 domain of the immunoglobulin molecule.

Amino Acid Sequence↗
PubMed1995

Inhibition of complement-mediated red cell lysis by immunoglobulins is dependent on the IG isotype and its C1 binding properties.

We have investigated the effect on complement activation of human immunoglobulins (Ig) using several therapeutic Ig preparations including two for intravenous use (IVIG), and various purified myeloma proteins. Ig inhibited lysis in a dose-dependent manner in the classical pathway assay whereas no alternative pathway inhibition was observed. The Fc part of the molecule was responsible for all the inhibitory effect. Purified IgG3 myeloma proteins were potent inhibitors whereas IgG1 inhibited to a lesser extent and IgG2 and IgG4 did not inhibit at all. Inhibition was obtained both when Ig was added to the solution and when it was coated onto a solid matrix. Analysis of the soluble and solid phase Ig after incubation revealed binding of C1q and activated C4 and C3 to the isotypes which inhibited lysis. Using selectively depleted sera and reconstitution with their respective purified components, efficient inhibition of lysis was seen when Ig was added prior to serum (C1), some inhibition was seen at the C4 level, whereas no effect was seen when Ig was added at the C9 level. We conclude that the complement-modulatory effect of Ig in vitro is isotype specific and dependent mainly on competitive C1 binding by the Ig molecule in the absence of antigen.

Animals↗
PubMed1995

Comparison among opsonic activity, antimeningococcal immunoglobulin G response, and serum bactericidal activity against meningococci in sera from vaccinees after immunization with a serogroup B outer membrane vesicle vaccine.

Opsonic activity in sera from 27 military recruits vaccinated with the Norwegian meningococcal serogroup B outer membrane vesicle vaccine was measured as respiratory burst with polymorphonuclear leukocytes as the effector cells and meningococci of the epidemic strain as the target. The results were compared with antimeningococcal IgG antibodies against an outer membrane vesicle coat in an enzyme-linked immunosorbent assay and with serum bactericidal activity. The vaccinees were immunized twice, with a 6-week interval between the two. The serum samples studied were collected at day zero, after 6 weeks, and after 12 weeks. Both serum bactericidal activity and respiratory burst were measured by adding external serum as the complement source. The results revealed a significant increase in specific IgG response, serum bactericidal activity, and respiratory burst after vaccination. We found a highly significant correlation between the responses in all three assays (P < 0.0001). The highest correlation was found between respiratory burst and antimeningococcal IgG response (r = 0.93). This result strongly indicates that respiratory burst is mediated almost exclusively by IgG antibodies. The correlation between antimeningococcal IgG response and serum bactericidal activity was slightly lower (r = 0.83). The correlation between respiratory burst and serum bactericidal activity was further reduced (r = 0.78), and some of the sera revealed a marked preference for only one of the activities. This result means that respiratory burst and serum bactericidal activity in part are induced by different mediators, and to obtain a more complete picture of the potential protective activity, both assays should be applied to survey a vaccine trial.

Adult↗
PubMed1994

One disulfide bond in front of the second heavy chain constant region is necessary and sufficient for effector functions of human IgG3 without a genetic hinge.

We have created four IgG3 mutants without a normal hinge region: (i) m0 without a genetic hinge; (ii) m0/C131S, where Cys-131 in m0 was mutated to Ser; (iii) m0/231C232 (formerly HM-1), where a Cys residue was inserted in m0 between Ala-231 and Pro-232; (iv) m0/C131S/231C232, which is a hybrid of m0/231C232 and m0/C131S. The wild-type IgG3 and all mutants bind 5-iodo-4-hydroxy-3-nitrophenacetyl groups. The wild type and mutants, m15 (with 15 aa in the hinge), m0/231C232, and m0/C131S/231C232, were all positive for complement-mediated lysis, antibody-dependent cellular cytotoxicity mediated by peripheral blood leukocytes, and phagocytosis by U937. m0/C131S/231C232 was only weakly positive and sometimes negative for respiratory burst activity mediated by peripheral blood neutrophils (polymorphonuclear leukocytes), whereas m15, m0/231C232, and wild-type IgG3 were strongly positive. The m0 and m0/C131S mutants were mainly negative for complement-mediated lysis, antibody-dependent cell-mediated cytotoxicity, and phagocytosis by U937 and polymorphonuclear leukocytes. The results indicate that a hinge spacer region is not necessary, but the correct alignment of the two second heavy chain constant regions in the IgG3 molecule by a minimum of one disulfide bond is necessary and sufficient for effector functions.

Amino Acid Sequence↗
PubMed1994

Human IgG isotype-specific amino acid residues affecting complement-mediated cell lysis and phagocytosis.

In this report we describe the construction of anti-5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) mouse/human immunoglobulin (Ig) G4 chimeric molecules with altered amino acid residues in the CH2 domain. Three mutants are described. Gln-268 is substituted by His in gamma 4 Q268H, Ser-331 is substituted by Pro in gamma 4 S331P, and in gamma 4 Q268H/S331P both residues are substituted. The ability of the mutant molecules to induce complement-mediated cell lysis (CML) and phagocytosis by Fc gamma RII- and Fc gamma RIII-bearing polymorphonuclear leukocytes (PMN) were measured. In CML, gamma 4 Q268H was inactive, but both gamma 4 S331P and gamma 4 Q268H/S331P were active provided that the antigenic density on the target cells was high. In phagocytosis mediated by PMN, the mutants gamma 4 S331P and gamma 4 Q268H/S331P were both active only when complement was introduced. gamma 4 Q268H was not active in phagocytosis under any conditions. We conclude that His-268 in human IgG molecules does not modulate CML activity or phagocytosis mediated by Fc gamma RII and/or Fc gamma RIII. Pro-331 rescues CML activity in IgG4 molecules when the epitope density on the target cells is high, but does not affect Fc gamma RII/Fc gamma RIII-mediated phagocytosis. In this manner the mutants gamma 4 S331P and gamma 4 Q268H/S331P mimic human IgG2. This could indicate a structural similarity between IgG2 and these mutant molecules that distinguish them from both IgG1 and IgG3.

Animals↗
PubMed1994

A comparison of human and murine monoclonal IgGs specific for the P1.7 PorA protein of Neisseria meningitidis.

Monoclonal human IgG SS269 reacts with Neisseria meningitidis expressing the P1.7 PorA protein and with linear peptides containing NGGAS, which accounts for the P1.7 specificity. Murine monoclonal antibody to P1.7 reacts with peptides containing the overlapping epitope, ASGQ. The human and murine antibodies have similar affinities. The low avidity human antibody is very inefficient at stimulating complement-mediated bactericidal killing while the high avidity murine antibody efficiently kills bacteria. However, efficient opsonophagocytosis was mediated even at low concentrations of the human antibody and in the absence of complement, suggesting that low avidity antibodies might be protective against disease.

Adult↗
PubMed1994

Opsonophagocytic activity induced by chimeric antibodies of the four human IgG subclasses with or without help from complement.

The opsonophagocytic activity of the four human IgG subclasses was studied using chimeric mouse-human antibodies with specificity for the hapten NIP. As target cells we used haptenized sheep red blood cells and N. meningitidis, labelled with different amounts of hapten. We used polymorphonuclear leucocytes (PMN) as effector cells to measure respiratory burst (RB), and U937 to measure phagocytosis/rosette formation. When the target cells were opsonized with antibody only, and PMN used as effector cells, IgG3 was highly efficient, while IgG1 revealed an intermediate activity and IgG2 and IgG4 were negative. The same pattern among the subclasses was obtained in the presence of complement source, when target cells with low hapten concentration were used. However, at high epitope concentration on the target cells, in the presence of complement source, IgG2 was highly active, while IgG4 was still negative or only slightly positive. When U937 were used as effector cells and complement was omitted, IgG1, IgG3 and IgG4 all revealed high phagocytic/rosette-forming activity, while IgG2 was negative. When the target cells were opsonized with antibody and complement, the phagocytic/rosette-forming activity was often suppressed. Our results reveal that all four human IgG subclasses possess opsonophagocytic capacity, but with different requirements concerning complement and Fc gamma Rs. They also enlighten us as to how IgG2 might perform its protective effect against harmful bacteria displaying high density of carbohydrate epitopes on their outside surface.

Animals↗
PubMed1993

Activation of complement by an IgG molecule without a genetic hinge.

The hinge region links the two Fab arms to the Fc portion of the IgG molecule. It mediates flexibility to the molecule and serves as a connecting structure between the two heavy chains. In addition it provides space between the Fab and Fc parts. All three properties have been proposed to be important for the ability of IgG to initiate complement activation leading to complement-mediated cell lysis (CML). Here we report the construction of a hinge-deleted mouse-human chimaeric IgG3 molecule with specificity for the hapten NIP (3-iodo-4-hydroxy-5-nitrophenacetyl), HM-1. HM-1 lacks the genetic hinge, but has an introduced cysteine between Ala 231 (EU numbering) and Pro 232 in the lower hinge encoded by the CH2 exon. The introduced cysteine forms a disulphide bond between the two heavy chains of the molecule. In CML, HM-1 shows a greater activity than IgG3 wild type. This is the first time an IgG molecule without a genetic hinge has been found to be active in CML. We conclude that the hinge functioning as a spacer is not a prerequisite for complement activation. Rather, its major role seems to be to connect the heavy chains to each other in the amino-terminal part of CH2. Because HM-1 is expected to have low Fab-Fc flexibility, this molecular feature is probably of no importance for complement activation.

Animals↗