Biomedical subjects

A J Caton

Publications and source records attributed to A J Caton.

At least 37 records · Page 2Linked to original sources

PubMed1991

Antibodies that are specific for a single amino acid interchange in a protein epitope use structurally distinct variable regions.

We have analyzed how the immune system generates antibodies that are specific for analogues of an epitope on the influenza virus hemagglutinin (HA) that differ solely by the presence of Asp or Gly at amino acid 225. Most antibodies induced in response to HA(Asp225) use one of a few closely related variable (V) region structures that are encoded by characteristic VH/Vk gene segment combinations. Remarkably, none of these VH/Vk combinations was induced in response to HA(Gly225). Instead of modifying the HA(Asp225)-specific V regions by junctional variation or somatic mutation to recognize the altered epitope, new VH/Vk combinations were used. The expression of unique VH/Vk combinations appears to confer exquisite specificity to the selection of HA-specific B cells from the pre-immune repertoire.

Amino Acid Sequence↗

PubMed1991

Many variable region genes are utilized in the antibody response of BALB/c mice to the influenza virus A/PR/8/34 hemagglutinin.

We have examined how many different H chain variable (VH) and kappa-chain variable (Vk) germ-line genes are used in the antibody response to the influenza virus A/PR/8/34 hemagglutinin (PR8 HA), and have assessed how the expression of individual VH and/or Vk genes contributes to the generation of specificity for the HA. A panel of 51 hybridoma antibodies that recognize two antigenic regions on the HA were compared for the sequence of their Ig H and L chain V regions. The hybridomas were obtained from 28 individual BALB/c mice that had been immunized with PR8 under a variety of primary and secondary response immunization protocols. The degree and pattern of sequence similarity suggests that 29 different VH genes drawn from seven different VH gene families, and 25 different Vk genes drawn from 12 different Vk gene families were used in this panel. Based on current estimates of the total numbers of VH and Vk genes in the mouse, this suggests that between 2.5 and 10% of the entire VH and Vk germ-line repertoires were used by these hybridomas. Despite this extensive diversity, some V genes were repetitively identified among these hybridomas, and were most often expressed in the context of specific VH/Vk combinations. Because antibodies that used identical VH/Vk combinations also usually displayed similar reactivity patterns with a panel of mutant viruses, this indicates that VH/Vk pairing can be important in establishing the specificity of antibodies for the HA.

Amino Acid Sequence↗

PubMed1991

Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes.

We have examined at the molecular level the CDR3 and adjacent regions in peripheral blood B lymphocytes of normal individuals. A total of 111 sequences (12-28 sequences from six individuals) were obtained after cloning of the polymerase chain reaction-amplified segments into plasmids or phage. The average length of the VDJ joining was 109 nucleotides, with a range from 79 to 151. Approximately 75% of the sequences were in frame when translated into amino acids. Among the JH segments, JH4 was found most frequently (in 52.5% of the sequences), and JH1 and JH2 segments the least frequently (approximately 1% of the clones). A polymorphic JH6 gene with a one-codon deletion accompanied by a base change was present in two of six patients. Preferential breakpoints were found for JH2, JH3, JH4, and JH5, although the breakpoints of JH6 were distributed more heterogenously. In approximately 90% of the cases, significant homology of the D regions with published D sequences was found. Preferential usage of a particular coding frame was observed in in-frame sequences utilizing DA, D21/9, and DM1 segments. However, in general, all coding frames of germline D genes were used to generate CDR3s. Eight sequences that have a DN1-like D sequence with two base changes at the same positions were identified, suggesting the likely existence of a new germ line D gene belonging to the DN families. Using probes specific for a particular CDR3, the frequency of a specific B cell clone in the peripheral blood of normal individuals was estimated to be at most as high as 1/20,000.

Amino Acid Sequence↗

PubMed1991

A B cell population that dominates the primary response to influenza virus hemagglutinin does not participate in the memory response.

The early primary B cell response of BALB/c mice to the influenza virus A/PR/8/34 (PR8) hemagglutinin (HA) is dominated by B cells that utilize a single V kappa gene in association with one of two closely related VH genes. We have used an anti-idiotypic reagent that recognizes a light chain-associated idiotope (23-1 Id) on these antibodies to follow their presence during the anti-HA response. Quantitation of 23-1Id+ antibodies at different time points during the anti-HA response indicates that the 23-1Id+ B cell response peaks early after primary immunization and is not re-induced by secondary challenge. Furthermore, 23-1Id+ titers in serum decay rapidly between the first and second week after immunization, and the HA-specific 23-1Id+ precursor B cell population does not significantly expand in the months following immunization. These results indicate that despite their predominance during the primary response, 23-Id+ B cells abruptly disappear from the response and do not mature into memory B cells.

Animals↗

PubMed1991

A new mouse T-cell receptor alpha chain variable region family.

Sequence analysis of the rearranged T-cell receptor alpha chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribed V alpha gene family. We have designated this family V alpha 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously described V alpha haplotypes: V alpha a, V alpha b, and V alpha c.

Amino Acid Sequence↗

PubMed1991

Identification of eight determinants in the hemagglutinin molecule of influenza virus A/PR/8/34 (H1N1) which are recognized by class II-restricted T cells from BALB/c mice.

Eight nonoverlapping regions of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8), which serve as recognition sites for class II-restricted T cells (TH) from BALB/c mice, have been identified in the form of 10- to 15-amino-acid-long synthetic peptides. These TH determinants are located between residues 110 to 313 of the HA1 polypeptide. From a total of 36 HA-specific TH clones and limiting-dilution cultures of independent clonal origins, 33 (90%) responded to stimulation with one of these peptides. The residual three TH clones appeared to recognize a single additional determinant on the HA1 polypeptide which could not be isolated, however, in the form of a stimulatory peptide. None of the motifs that have been proposed to typify TH determinants were displayed by more than half of these recognition sites. Most unexpected was the finding that none of the TH determinants was located in the ectodomain of the HA2 polypeptide that makes up roughly one-third of the HA molecule. Possible reasons for the preferential recognition of HA1 as opposed to HA2 by TH are discussed.

Amino Acid Sequence↗

PubMed1990

Structure-function relationships among highly diverse T cells that recognize a determinant from influenza virus hemagglutinin.

We have analyzed the structural and genetic basis for T cell recognition of the complex formed between antigen and class II products of the major histocompatibility complex by performing sequence analysis of T cell receptors (TCRs) induced in response to the helper T cell site 1 of the influenza virus hemagglutinin. The results demonstrate, first, that structurally highly diverse TCRs can be utilized in recognition of the same antigen/I-Ed complex: 12 of 13 TCRs utilize unique V alpha/V beta gene segment combinations, suggesting that approximately 70 different V alpha/V beta combinations are available to BALB/c mice in response to this determinant. Second, comparison of these sequences with the ability of each hybridoma to recognize a panel of peptide analogues suggests that alpha and beta chains of these TCRs frequently determine specificity for the NH2-terminal and the COOH terminal portions, respectively, of the site 1 determinant.

Amino Acid Sequence↗

PubMed1990

A set of closely related antibodies dominates the primary antibody response to the antigenic site CB of the A/PR/8/34 influenza virus hemagglutinin.

Approximately 50% of the primary antibody response of BALB/c mice to the A/PR/8/34 influenza virus hemagglutinin is directed to the Cb site, one of the four major antigenic regions of the molecule. To determine the structural basis of the anti-Cb site response, we have examined the paratypic and genetic diversity exhibited by a panel of 24 primary and 4 secondary response mAb specific for this antigenic region. Reactivity pattern analysis demonstrated 20 distinct fine specificities among these antibodies, and V region gene sequence analysis showed that they are encoded by 17 different VH gene segments from 6 VH gene families and 14 different VK gene segments from 6 VK gene groups. Despite this overall diversity, many of the antibodies can be placed in a limited number of sets based on the shared expression of VH and/or VK genes. One set contains antibodies encoded by a single gene of the VK4/5 group in combination with one of two closely related genes from the J558 VH family. This set accounts for half of the Cb site-specific primary response hybridomas, indicating that the representation of the various anti-Cb site B cell specificities during the primary response to A/PR/8/34 influenza virus is not uniform. The preferential participation of B cells expressing this VH/VK combination is largely responsible for the dominance of anti-Cb site antibodies in the primary anti-hemagglutinin response.

Amino Acid Sequence↗

PubMed1990

A single pre-B cell can give rise to antigen-specific B cells that utilize distinct immunoglobulin gene rearrangements.

A group of hybridomas that express antibodies with related specificities for the influenza virus hemagglutinin (HA), that represent B cells that were the clonal progeny of a single pre-B cell, and that utilized distinct L chain gene rearrangements have been characterized. The clonal relationship was established by the sharing of H chain gene rearrangements at both the productive and the nonproductive alleles. Among these hybridomas, one group had rearranged only one of its kappa alleles, having joined a V kappa 24 gene to the J kappa 2 gene segment. The other group utilized the same V kappa 24 gene segment in productive rearrangement to the J kappa 5 gene segment, and shared an aberrant rearrangements among members of the same B cell clone can normally occur, and can contribute to the generation and diversification of the immune repertoire that is available for the recognition of foreign antigens. Mechanisms by which the distinct rearrangements expressed by the hybridomas might have been generated are discussed.

Alleles↗

PubMed1990

Identical D region sequences expressed by murine monoclonal antibodies specific for a human tumor-associated antigen.

Sequence analysis has revealed significant structural similarities among murine mAb specific for the human tumor-associated Ag GA733. Antibodies generated in independent immunizations, either with Ag-positive tumor cells or with anti-idiotypic antibodies produced by immunization of goats with a GA733-specific antibody, all use members of the same gene families; remarkably, they also express identical amino acid sequences in their H chain CDR3. Inasmuch as this region normally exhibits considerable sequence variability, the identity displayed by the antibodies indicates a requirement for this particular sequence in the generation of their specificity for the GA733 antigen. Moreover, this homology suggests that the antibodies recognize a common determinant on the Ag, and that the polyclonal anti-idiotypic antibodies can functionally mimic the Ag at the level of the structure of the Ag-specific antibody that is induced.

Amino Acid Sequence↗

PubMed1990

A new Igk-V gene family in the mouse.

We have identified and characterized a novel mouse Igk-V gene family, which we have designated Igk-V34. Southern hybridization and nucleotide sequence analysis indicate that this family is comprised of either one or two members in mice of different Igk haplotypes. The gene family members share between 95% and 98% sequence similarity, indicating that they diverged only recently during the evolution of the Igk locus. Sequence relationships between members of this family are discussed.

Amino Acid Sequence↗

PubMed1990

Influenza virus hemagglutinin-specific antibodies isolated from a combinatorial expression library are closely related to the immune response of the donor.

Antibodies specific for the influenza virus hemagglutinin have been isolated from a combinatorial expression library generated using mRNA obtained from an immunized donor mouse. Sequence analysis indicates that the antibody heavy chain variable regions were derived from members of an expanded hemagglutinin-specific B-cell clone, in conjunction with one of two light chain variable regions. Moreover, the most frequently identified heavy chain variable/light chain variable combination is extremely similar to a heavy chain variable/light chain variable combination that has previously been identified among hemagglutinin-specific hybridoma antibodies. The results, therefore, demonstrate that these antibodies bear a close relationship to the immune status of the donor mouse and suggest that simple adaptations of this procedure might allow evaluation of the immune responses of species, such as man, in which conventional hybridoma techniques have to date proven ineffective.

Amino Acid Sequence↗

PubMed1989

Detection of minimal disease in hematopoietic malignancies of the B-cell lineage by using third-complementarity-determining region (CDR-III)-specific probes.

Approximately 80% of hematopoietic malignancies of the B-cell lineage carry only one or two immunoglobulin heavy chain gene rearrangements indicating their clonal origin. These rearrangements due to the recombination of various variable, diversity, and joining regions of the heavy-chain gene segments during B-cell commitment result in a region called complementarity-determining region III (CDR-III). This region, which encompasses the diversity region of the heavy-chain segment, because of extensive somatic mutations, provides a DNA-encoded signature specific for each B-cell clone. CDR-III sequences were obtained from DNA of pre-B-cell acute lymphoblastic leukemia by using suitable primers and the polymerase chain reaction. The sequences were used to generate diagnostic probes that hybridized only to the amplified CDR-III of leukemic cells from which the sequences were derived. With these probes, leukemic cells could be detected when diluted 1:10,000 with other cells. By cloning the amplified CDR-III into recombinant libraries residual leukemic cells were accurately quantitated in bone-marrow samples from repeated relapses and remissions in one case of acute lymphoblastic leukemia. During a clinical remission lasting greater than 7 mo, malignant cells were present in marrow at greater than 1 per 1000 cells. These findings indicate that custom-made diagnostic probes will be useful in accurate quantitation of malignant cells in acute lymphoblastic leukemia patients in clinical remission and will allow investigation of the biological significance of low or high numbers of residual leukemic cells in evolution of that disease.

Alleles↗

PubMed1988

Hemagglutinin polymorphism as the basis for low- and high-yield phenotypes of swine influenza virus.

Single amino acid substitutions at the rim of the receptor binding site of the hemagglutinin molecule of swine influenza virus markedly influence the replicative capacity of the virus in chicken embryos, Madin-Darby canine kidney cells (MDCK), and swine as well as its antigenic phenotype. Mutants of low-yield (L) phenotype replicate poorly in chicken embryos and produce small plaques in MDCK cells but are highly infective for swine. Such mutants have lysine at position 153 and glycine at position 155 of the hemagglutinin (residues 156 and 158 in the H3 model). High-yield (H) mutants have the converse replicative characteristics and can be antigenically distinguished from L mutants (and from each other) based on their differential reactivity with two monoclonal antibodies, 9C8 and Sa-13. H mutants differ from L mutants in that the H mutants express glutamic acid at either position 153 or 155. L and H mutants act in an allelic fashion in effecting predictable one-step adaptation to different hosts. Selection for replication (e.g., high-yielding) phenotype results in concordant pleiotropic change in antigenic phenotype and in genotype. Conversely, immunoselection leads to change in replicative phenotype. Although the mechanism by which these mutations affect viral replication has not yet been defined, they may reflect differences in the affinity of each mutant for different host receptors.

Animals↗

PubMed1986

The antigenicity and evolution of influenza H1 haemagglutinin, from 1950-1957 and 1977-1983: two pathways from one gene.

Nucleotide sequence analysis of the region of the haemagglutinin gene coding for the HA1 domain of the protein was performed on 19 human influenza A strains of H1 subtype representative of the two epidemic periods from 1977-1983 and from 1950-1957. The amino acid changes relative to A/USSR/90/77 are summarised and are consistent with the view that variation in these field strains involves changes largely at the Sb and Ca antigenic sites previously characterised in laboratory mutants of the haemagglutinin of influenza A/PR/8/34. The Sa and Cb sites are less variant and are probably masked by carbohydrate side chains. We discuss the significance of other amino acid changes which do not correspond to previously defined antigenic sites. We also define the "mainstream" amino acid changes characteristic of the divergent evolutionary pathways of the 1950-1957 and 1977-1983 periods and note that the rate of evolution is faster in the earlier period.

Antigens, Viral↗

PubMed1986

Structural and functional implications of a restricted antibody response to a defined antigenic region on the influenza virus hemagglutinin.

A group of hybridoma antibodies that recognize structurally overlapping epitopes on the influenza virus hemagglutinin have been analyzed for the sequence of their immunoglobulin heavy and light chain variable regions. All VH regions derive from the same gene family, and only two Vk genes, from different families, are involved. The repetitive and restricted use of these variable region genes indicates that considerable structural requirements influence the generation of antibodies specific for this region of the hemagglutinin. The degree of amino acid variability which is permissive for interaction with this region suggests that two thirds of the possible replacement mutations may abolish either antibody function or specificity. Analysis of the somatic mutation which occurred in the individual antibodies indicates that the light chains acquired replacement mutations at the rate predicted for random mutation. The heavy chains, however, accumulated a 3-fold excess of replacement mutations over that predicted for random accumulation, correlating with the dominant role they apparently play in determining fine differences in the specificity of these antibodies. The effect of somatic mutation on the clonal amplification and diversification of these B cell lineages is discussed.

Animals↗

PubMed1986

Host cell-mediated selection of a mutant influenza A virus that has lost a complex oligosaccharide from the tip of the hemagglutinin.

During serial passage in Madin-Darby bovine kidney (MDBK) cells, a substrain of influenza virus A/WSN is lost from the population and is replaced by a mutant virus with altered host cell binding properties. This selection does not occur during growth in chicken embryo fibroblasts (CEF). It occurs during growth in MDBK cells because the parental virus produced by these cells has a dramatically reduced affinity for cellular receptors [Crecelius, D.M., Deom, C. M. & Schulze, I. T. (1984) Virology 139, 164-177]. We have now compared the hemagglutinin (HA) subunits, HA1 and HA2, of the parent and mutant viruses by NaDodSO4/PAGE and have found that when the viruses are grown in either host cell the HA1 subunit of the mutant is smaller than that of the parent virus. The nonglycosylated HAs, made in the presence of tunicamycin, have the same apparent molecular weight, indicating that the HA1 subunit of the mutant virus contains less carbohydrate than that of the parent. This reduction in carbohydrate content was observed with 11 independently derived mutants that had been selected by growth in MDBK cells. The nucleotide sequence of the HA gene of the parent and mutant viruses indicates that there are five potential glycosylation sites on the parent HA1 subunit and four on the mutant and that the mutation responsible for this difference is a single base change that eliminates the glycosylation site at amino acid 125 of the parent HA1 subunit. Treatment of the parent and mutant HAs from both cell sources with endo-beta-N-acetylglucosaminidases F and H showed that the HA1 of the parent virus has four complex and one high-mannose oligosaccharides, whereas that of the mutant virus has three complex and one high-mannose oligosaccharides. Thus, all of the potential sites on both HA1 subunits are glycosylated. We conclude that the oligosaccharide attached to amino acid 125 of the parent HA by MDBK cells can reduce the affinity of the virus for cellular receptors and that the mutant virus has a higher affinity than the parent because the mutant HA is not glycosylated at that site. Since amino acid 125 of the parent HA is glycosylated by both CEF and MDBK cells, we further conclude that the host-determined structure of the oligosaccharide at that site affects the affinity of the parent virus for cellular receptors and, thereby, determines whether the mutant virus will have a growth advantage.

Amino Acid Sequence↗

PubMed1986

Selection of influenza A virus adsorptive mutants by growth in the presence of a mixture of monoclonal antihemagglutinin antibodies.

The influenza virus hemagglutinin contains four major regions that are recognized by antibodies able to neutralize viral infectivity. To investigate the effect of an antibody response directed against each of these sites on viral evolution, influenza virus A/PR/8/34 (H1N1) was grown in allantois-on-shell cultures in the presence of a mixture of monoclonal antihemagglutinin antibodies. This selection mixture contained antibodies (two or three antibodies per antigenic site) whose concentrations were adjusted to achieve equal neutralization titers against each of the four antigenic sites. By varying the ratio of input virus to selection mixture concentration, we observed that variant viruses emerged under conditions of partial neutralization. Each of the four variants characterized in detail differed from the parental virus in its interaction with cellular receptors and exhibited minimal changes in antigenicity. Thus, these variants were virtually indistinguishable from wild-type viruses, as assessed by the binding of 103 monoclonal antihemagglutinin antibodies in an indirect radioimmunoassay. Despite this, many of the same antibodies demonstrated decreased titers to the variants in hemagglutination inhibition tests. The magnitude of the differences depended on the indicator erythrocytes used (much greater differences were detected with chicken erythrocytes than with human erythrocytes). Hemagglutination mediated by the variants was more resistant to neuraminidase treatment of erythrocytes than hemagglutination mediated by the parental virus. These findings are consistent with the idea that the variants were initially selected by virtue of their increased avidity for host cell receptors. Sequencing of viral RNA revealed that each of the variants differed from the parental virus by a single amino acid alteration in its HA1 subunit. Two of the changes were close to the proposed receptor binding site on hemagglutinin and could directly alter receptor binding, while a third was located near the trimer interface and may have increased receptor binding by altering monomer-monomer interactions.

Adhesiveness↗