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T M Buchanan

Publications and source records attributed to T M Buchanan.

At least 19 recordsLinked to original sources
PubMed1992

Identification of overlapping HLA class I-restricted cytotoxic T cell epitopes in a conserved region of the human immunodeficiency virus type 1 envelope glycoprotein: definition of minimum epitopes and analysis of the effects of sequence variation.

Although the immunologic basis of protective immunity in human immunodeficiency virus type 1 (HIV-1) infection has not yet been defined, virus-specific cytotoxic T lymphocytes (CTL) are likely to be an important host defense and may be a critical feature of an effective vaccine. These observations, along with the inclusion of the HIV-1 envelope in the majority of vaccine candidates presently in clinical trials, underscore the importance of the precise characterization of the cellular immune responses to this protein. Although humoral immune responses to the envelope protein have been extensively characterized, relatively little information is available regarding the envelope epitopes recognized by virus-specific CTL and the effects of sequence variation within these epitopes. Here we report the identification of two overlapping CTL epitopes in a highly conserved region of the HIV-1 transmembrane envelope protein, gp41, using CTL clones derived from two seropositive subjects. An eight-amino acid peptide was defined as the minimum epitope recognized by HLA-B8-restricted CTL derived from one subject, and in a second subject, an overlapping nine-amino acid peptide was identified as the minimal epitope for HLA-B14-restricted CTL clones. Selected single amino acid substitutions representing those found in naturally occurring HIV-1 isolates resulted in partial to complete loss of recognition of these epitopes. These data indicate the presence of a highly conserved region in the HIV-1 envelope glycoprotein that is immunogenic for CTL responses. In addition, they suggest that natural sequence variation may lead to escape from immune detection by HIV-1-specific CTL. Since the region containing these epitopes has been previously shown to contain an immunodominant B cell epitope and also overlaps with a major histocompatibility complex class II T cell epitope recognized by CD4+ CTL from HIV-1 rgp160 vaccine recipients, it may be particularly important for HIV-1 vaccine development. Finally, the identification of minimal CTL epitopes presented by class I HLA molecules should facilitate the definition of allele-specific motifs.

Amino Acid Sequence↗
PubMed1991

HIV-1 gag-specific cytotoxic T lymphocytes recognize multiple highly conserved epitopes. Fine specificity of the gag-specific response defined by using unstimulated peripheral blood mononuclear cells and cloned effector cells.

CTL directed at the highly conserved HIV-1 gag protein have been described in HIV-1 seropositive persons and may be an important host defense against this retrovirus. Presently only limited data are available regarding the specific epitopes recognized by these CTL. In this study, we have performed a detailed examination of the gag-specific CTL response in three HIV-1 seropositive subjects, using both unstimulated PBMC and cloned CTL. Lysis of gag-expressing targets was found to be mediated by CD3+CD8+ lymphocytes and restricted by class I Ag. Multiple class I Ag were found to restrict gag epitopes in each subject studied, with as many as three of these Ag involved in presenting gag CTL epitopes in a single subject. The majority of gag-specific CTL activity was found to be directed against epitopes in the p24 subunit of the gag protein, with at least seven different HLA class I-restricted CTL p24 epitopes identified in these three subjects. Less CTL activity was directed against p17 subunit of gag and two CTL epitopes were identified in this protein. Although as many as four different epitopes in gag were recognized using CTL from a single subject, none of the epitopes was recognized by CTL from more than one subject. Analysis of gag epitope recognition using cloned CTL demonstrated heterogeneity and specificity not appreciated using unstimulated PBMC. The identification of multiple relatively conserved epitopes in the HIV-1 gag protein and the heterogeneity of CTL responses to this protein may have important implications for vaccine development and our understanding of AIDS pathogenesis.

Amino Acid Sequence↗
PubMed1991

T cell activation by mycobacterial antigens in inflammatory synovitis.

To define which mycobacterial antigens were responsible for the activation of synovial fluid T lymphocytes, acetone-precipitated Mycobacterium tuberculosis (AP-MT) antigens were separated into five fractions following polyacrylamide gel electrophoresis and added to the mononuclear cell cultures of patients with inflammatory synovitis. Fractions 2 (50 to 70 kDa) and 5 (less than 28 kDa) resulted in significantly more proliferation than that of fractions 1, 3, and 4. The response to a purified mycobacterial 65-kDa heat shock protein (hsp), which migrated in fraction 2, was highly correlated (r = 0.89, P less than 0.001) with the response to the crude AP-MT. The proliferative response to a different hsp. the Escherichia coli DnaK, by synovial fluid lymphocytes was marginal. Analysis of the synovial fluid T cell response to mycobacterial culture filtrates by T cell Western blotting revealed dominant responses to antigen(s) in the range of 31 to 21 kDa in each responding patient, although no other consistent pattern of T cell activation was noted. Three lines of evidence suggested that the response to the low molecular weight fractions was directed against degradation fragments of the 65-kDa protein. These observations suggest that the activation of T lymphocytes obtained from inflammatory synovial fluids by crude mycobacterial antigens was due in large part to recognition of the 65-kDa mycobacterial hsp.

Antigens, Bacterial↗
PubMed1989

Monoclonal antibodies to protein I for serotyping of Neisseria gonorrhoeae: correlation of serotype with bactericidal activity.

Seven monoclonal antibodies have been used for the serotyping of one hundred Neisseria gonorrhoeae wild strains, randomly selected from nine U.S. cities, and seven serotype reference strains by the co-agglutination method. As determined by gel-immunoradioassay, the monoclonal antibodies recognized the protein I trimer of a single or a limited subset of serotype reference strains. All but three strains were typable by one or two of the antibodies. The most common serotypes were 1.3 (26%), 1 (20%), 5 (17%), 5.7 (11%) and 9 (10%). To correlate typing results with ability for killing of these antibodies, susceptibility of typed and non-typed strains to killing was studied. Susceptibility was significantly associated with typing by the serotype 7 (p = 0.011) and serotype 9 (p = 0.033) specific monoclonal antibodies. Reaction of antibodies recognizing epitopes on the protein IB molecule with a given strain predicted in an average of 43% of strains (49% of strains of serotype 5, 62% of serotype 7, 29% of serotype 8, and 33% of serotype 9) its susceptibility to killing by the typing antibodies. In contrast, only 15% of the strains (15% of strains of serotype 1 and 15% of serotype 3) were killed by their typing antibodies, recognizing epitopes on the protein IA molecule. These monoclonal antibodies might prove to be important for the isolation and structural characterization of epitopes responsible for susceptibility of the gonococcus to killing and thus for the development of a vaccine against invasive gonococcal disease.

Agglutination Tests↗
PubMed1989

Interaction of non-piliated Neisseria gonorrhoeae strain 7122 and protein IA with an epithelial cell monolayer.

Studies with [14C] uracil-labelled bacteria revealed that the interaction of Neisseria gonorrhoeae with epithelial cells occurred in a time-dependent reaction which is slightly pH-dependent and optimal at pH 6.5. Immunofluorescence tests and immunoelectron microscopy of ultrathin sections confirmed the attachment of these bacteria to the epithelial cell membrane. The interaction of purified protein I with epithelial cells was time-dependent and reached equilibrium after four hours as shown by tracer experiments with 125I-labeled protein I. Cleavage experiments with trypsin followed by SDS-PAGE and autoradiography indicated that protein I (labeled with 125I) was associated with the membrane of the epithelial cells and only partly accessible by trypsin after its interaction with these mammalian cells. Immunofluorescence tests as well as immunoelectron microscopy with the monoclonal antibody G7A2C and gold-labeled protein A confirmed a dense association pattern of protein I with the cell monolayer.

Antibodies, Monoclonal↗
PubMed1989

Use of an antigen-capture assay for characterisation of monoclonal antibodies to mycobacterial lipoarabinomannan.

Monoclonal antibodies directed to six separate antigen molecules of Mycobacterium leprae have been tested in an antigen-capture assay based on combined use of polyclonal ("capture") and monoclonal ("detector") antibody reagents. This approach provides a potentially versatile, sensitive and specific assay for detection and relative quantitation of M. leprae antigens. Characterisation of monoclonal antibodies to mycobacterial lipoarabinomannan (LAM-B) by the antigen-capture assay indicates that some of the antigenic determinants present on LAM-B from M. leprae may be either absent altogether or present at much lower concentrations on the corresponding LAM-B structure form M. tuberculosis.

Antibodies, Monoclonal↗
PubMed1988

A Mycobacterium leprae-specific human T cell epitope cross-reactive with an HLA-DR2 peptide.

Mycobacterium leprae induces T cell reactivity and protective immunity in the majority of exposed individuals, but the minority that develop leprosy exhibit various types of immunopathology. Thus, the definition of epitopes on M. leprae antigens that are recognized by T cells from different individuals might result in the development of an effective vaccine against leprosy. A sequence from the 65-kD protein of this organism was recognized by two HLA-DR2-restricted, M. leprae-specific helper T cell clones that were derived from a tuberculoid leprosy patient. Synthetic peptides were used to define this epitope as Leu-Gln-Ala-Ala-Pro-Ala-Leu-Asp-Lys-Leu. A similar peptide that was derived from the third hypervariable region of the HLA-DR2 chain, Glu-Gln-Ala-Arg-Ala-Ala-Val-Asp-Thr-Tyr, also activated the same clones. The unexpected cross-reactivity of this M. leprae-specific DR2-restricted T cell epitope with a DR2 peptide may have to be considered in the design of subunit vaccines against leprosy.

Amino Acid Sequence↗
PubMed1988

Exact definition of species-specific and cross-reactive epitopes of the 65-kilodalton protein of Mycobacterium leprae using synthetic peptides.

With the use of solid phase synthesis of peptides corresponding to major and minor peaks in a Hopp-Woods hydrophilicity plot, the epitopes for 10 of 14 known different mAb to the Mycobacterium leprae 65-kDa protein, a prominent T and B cell Ag of this bacillus, have been located in the primary structure. Five epitopes have been precisely mapped by using the synthetic peptides in inhibition ELISA experiments, and five others have been located on peptides of 22 amino acids or less in length. The epitope of an important species-specific antibody, IIIE9, which may be useful for seriodiagnosis of leprosy, appears to be distinguished from the epitope of the antibody IVD2, widely cross-reactive among mycobacteria, not by its sequence, but only by its critical residues. All epitopes studied appear continuous insofar as can be determined by this approach.

Amino Acid Sequence↗
PubMed1988

Surface immunolabeling of Neisseria gonorrhoeae employing monoclonal antibodies to proteins IA and IB.

The technique of immunomarking intact bacteria with monoclonal antibodies (MAb) and gold-labeled anti-IgG provides a rapid and reliable method for localization of surface-exposed antigens. Cell suspensions of Neisseria gonorrhoeae were incubated with monoclonal antibodies directed against different epitopes of protein I as shown by ELISA inhibition test (unpublished data). The bound IgG molecules were detected with gold labeled anti-mouse IgG or protein A. MAbs against various epitopes of protein I showed differences in their reaction pattern with intact bacteria. We compared the results achieved by the gold immunomarking technique with coagglutination (CoA) and immunofluorescence (IF) tests. The results of the three methods correlated for some antibodies and were ambiguous for other MAbs. The advantage of the immunogold method over immunofluorescence and coagglutination is that distribution and localization of antigen on the surface of gonococci can be exactly determined using electron microscopy.

Agglutination Tests↗
PubMed1987

Protein I serotype of serum-resistant versus serum-sensitive Neisseria gonorrhoeae strains.

In order to characterize serum-resistant and serum-sensitive strains of N. gonorrhoeae, the protein I serotype, auxotype, and penicillin susceptibility of 128 strains were tested. Sensitivity to the complement-dependent bactericidal activity of normal human serum was highly associated with protein I serotype (p less than 0.001). Thus 85% of serotype 1-3 strains were serum-resistant, whereas 86% of serotype 8 strains and all strains with serotypes 8 + 9 or 9 were serum-sensitive. Serum-resistance or sensitivity for a given serotype was independent of auxotype. The susceptibility to penicillin within the serotypes 1-3 was significantly associated with auxotype (p = 0.0016); all AHU- (requirement for arginine, hypoxanthine and uracil) strains had MICs of penicillin of 0.04 microgram/ml or less and were serotypes 1-3. Among the non-AHU-strains, serotype 9 was significantly more penicillin susceptible than the other serotypes (p less than 0.003).

Blood Bactericidal Activity↗
PubMed1987

Most Mycobacterium leprae carbohydrate-reactive monoclonal antibodies are directed to lipoarabinomannan.

Each of more than 30 monoclonal antibodies that had been raised against Mycobacterium leprae and previously classified as reactive with carbohydrate was shown to be directed against lipoarabinomannan, a prominent, highly pervasive, myo-inositol-phosphate-containing, cross-reactive antigen within the leprosy bacillus. Some of the antibodies preferentially bound to the lipopolysaccharide of M. leprae rather than to that of Mycobacterium tuberculosis, suggesting the presence of distinguishing structural features. The presence of alkali-labile inositol 1-phosphate in the lipopolysaccharide from M. tuberculosis and its apparent absence from the M. leprae product may account for the difference.

Antibodies, Monoclonal↗
PubMed1987

Characterization of antibody-reactive epitopes on the 65-kilodalton protein of Mycobacterium leprae.

Twenty-three monoclonal antibodies (MAbs) prepared in seven different laboratories were studied, all of which recognized the 65-kilodalton (kDa) protein of Mycobacterium leprae as determined by Western blotting or gel radioimmunoassay or both. Fourteen of the MAbs recognized different epitopes, as evaluated by cross-competition studies using radiolabeled MAb and unlabeled inhibitors; the species specificity of these epitopes was defined by nitrocellulose dot blot immunoassays with bacterial sonic extract antigen preparations from 23 species of mycobacteria. Each of the 14 distinct MAbs recognized a 65-kDa protein produced by a lysogenized Escherichia coli Y1089 host containing cloned rDNA which included the gene for the M. leprae 65-kDa protein. Of the 14 distinct MAbs, 1 recognized an epitope found only on M. leprae, and the others recognized epitopes present on as few as 8 or as many as all 23 of the mycobacterial species studied. Identification of these distinct 65-kDa protein epitopes and use of the MAbs which recognize them should assist future structural studies of this protein and characterization of the T-cell reactive and serodiagnostically useful portions of the molecule.

Antibodies, Monoclonal↗
PubMed1987

Probing the surface of Neisseria gonorrhoeae: simultaneous localization of protein I and H.8 antigens.

Gonococcal outer membrane protein I and the neisserial antigen H.8 are being investigated for inclusion in a gonococcal vaccine. To determine the distribution of immunoaccessible protein I and H.8 molecules on the surface of viable gonococci and to approximate the accessibility of these antigens to vaccine-elicited antibodies, immunologic probes composed of protein I- and H.8-specific antibodies linked to gold spheres were developed. When whole gonococci were exposed to the protein I and H.8 immunologic probes and examined by transmission electron microscopy, gold spheres clearly marked the surface of some of the gonococci, but not the surface of other gonococci from the same culture. The immunologic accessibility of gonococcal protein I or neisserial H.8 varied among gonococci. This diversity may affect the efficacy of a vaccine composed of these surface antigens.

Antibodies, Monoclonal↗
PubMed1986

ELISA detection of IgM antibodies against phenolic glycolipid-I in the management of leprosy: a comparison between laboratories.

IgM antibodies to the phenolic glycolipid-I (PGL-I) antigen of Mycobacterium leprae were detected by different ELISA techniques in three laboratories (in New York, Colorado, Seattle, U.S.A.). The agreement on seropositivity and overall correlation between techniques was excellent. A positive linear correlation between the bacterial index (BI) and anti-PGL-I IgM, previously reported by the New York laboratory, was detected by all techniques. The role of erythema nodosum leprosum in decreasing the relationship of BI versus anti-PGL-I IgM was seen by the New York laboratory with sera diluted 1:20 and ABTS substrate solution and by the Colorado laboratory but not by New York with sera at 1:300 and OPD substrate or by the Seattle laboratory.

Antibodies, Bacterial↗