Vaccines for the Third World.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
B R Bloom
Publications and source records attributed to B R Bloom.
Lymphocytes bearing antigen-specific gamma delta T-cell receptors accumulate in human infectious disease lesions.
The majority of T cells bear the T-cell receptor (TCR) alpha beta complex which recognizes foreign antigen peptides only in the context of self major histocompatibility complex (MHC) molecules. Such T cells function in a variety of effector roles and secrete cytokines that mediate the activation and differentiation of other cells in the immune system. Recently, a small subpopulation T cells was found to bear a distinct TCR composed of gamma and delta subunits. In man, TCR gamma delta+ cells are distributed as approximately 5 per cent of the CD3+ cells in all organized lymphoid organs as well as in the skin- and gut-associated lymphoid tissues. Although a limited number of germ-line genes encode the TCR gamma and delta subunits, extensive junctional variation particularly in the delta gene, results in unprecedented diversity for this receptor. The nature of the specificity and immunological functions of these T cells remains enigmatic. We report here that in contrast to the normal low frequency of gamma delta-bearing cells in lymphoid tissues, peripheral blood, or normal skin, the frequency is increased five to eightfold in particular granulomatous reactions of leprosy. TCR gamma delta+ lymphocyte lines from these leprosy skin lesions proliferate in vitro specifically to mycobacterial antigens. This reactivity to foreign antigens appears to require presentation in the context of self-molecules. Moreover, culture supernatants from activated gamma delta T lymphocytes induce adhesion and aggregation of bone-marrow monocytes in the presence of granulocyte monocyte-colony stimulating factor (CSF), suggesting that products of gamma delta-bearing T cells may play a role in the immune response, possibly by stimulating granuloma formation.
Epitopes↗
Analysis of naturally occurring delayed-type hypersensitivity reactions in leprosy by in situ hybridization.
Analysis of tissue lesions of the major reactional states of leprosy was undertaken to study the immune mechanisms underlying regulation of cell-mediated immunity and delayed-type hypersensitivity (DTH) in man. In situ hybridization hybridization of reversal reaction biopsy specimens for INF-gamma mRNA expression revealed a 10-fold increase in specific mRNA-containing cells over that observed in unresponsive lepromatous patients. Expression of huHF serine esterase, a marker for T cytotoxic cells, were fourfold increased in reversal reaction and tuberculoid lesions above that detected in unresponsive lepromatous individuals. Immunohistology of reversal reactions confirmed a selective increase of Th and T cytotoxic cells in the cellular immune response. Of interest, the microanatomic location of these serine esterase mRNA-containing cells was identical to the distribution of CD4+ cells. Analysis of erythema nodosum leprosum (ENL) lesions revealed differences in the underlying immune processes in comparison with reversal reaction lesions. Although phenotypic Th cells predominated in ENL lesions, IFN-gamma and serine esterase gene expression were markedly reduced. We suggest that reversal reactions represent a hyperimmune DTH response characterized by a selective increase of CD4+ IFN-gamma producing cells and T cytotoxic cells, which result in the clearing of bacilli and concomitant tissue damage. In contrast, ENL reactions may be viewed as a transient diminution of Ts cells and activity leading to a partial and transient augmentation in cell-mediated immunity, perhaps sufficient to result in antibody and immune complex formation, but insufficient to clear bacilli from lesions.
Esterases↗
Isolation and characterization of the highly immunogenic cell wall-associated protein of Mycobacterium leprae.
In a recent study, we demonstrated that certain reactivities crucial to the immune response in leprosy are due to protein associated with the cell wall peptidoglycan "core" of Mycobacterium leprae. We now describe a primary method for the isolation of a highly immunogenic, large molecular-size, cell wall protein (CW-P) complex from M. leprae, freed of soluble proteins, bound mycolates, arabinogalactan, and much of the peptidoglycan. The complex is of apparent relative molecular size 2 x 10(6) to 20 x 10(6) Da, is distinguished by a high content of Ala, Gly, Leu, Asx, and Glx, and some peptidoglycan, and represents up to 7% of the bacterial mass. It is stable to a variety of dissociation and reductive processes and, in accord with its size, is not resolvable by polyacrylamide gel electrophoresis. The mAb to the CW-P complex also react with the heat shock 65-kDa protein of M. leprae. Conversely, antibodies that recognize internal epitopes within the polypeptide chain of the heat shock protein also react with CW-P; however, antibodies that recognize the N and C termini of the 65-kDa protein fail to react with CW-P, and some anti-CW-P mAb do not recognize any of the soluble proteins of M. leprae. Alternate methods to derive the large peptidoglycan-associated protein result in lower yield and less of the associated heat shock protein, implying that the 65-kDa protein may not be crucial to the immunogenicity of the complex. In an accompanying paper, we demonstrate that T cell clones raised to CW-P also selectively recognize soluble proteins, primarily of 7-kDa and 16-kDa size. Thus, the image of the CW-P complex of M. leprae is of a few immunoreactive polypeptides in avid association with a modicum of peptidoglycan to which the 65-kDa polypeptide may be variably attached, perhaps due to involvement in assembly of the complex.
Amino Acids↗
Characterization of Mycobacterium leprae cell wall-associated proteins with the use of T lymphocyte clones.
Development of a vaccine against leprosy depends on the identification of Ag that stimulate cell-mediated immune responses. We have previously demonstrated that cell wall proteins of Mycobacterium leprae are highly immunogenic. By using human cell wall-specific T cell clones we have begun to characterize soluble proteins that integrate into the cell wall skeleton. T cells from leprosy lesions were expanded with IL-2 in vitro yet retained specificity to Ag of the insoluble cell wall core (CWC) in vitro, indicating that T cells had been activated by CWC Ag in vivo. A cell wall protein-peptidoglycan complex and cell wall protein preparations lacking carbohydrates and lipids from CWC retained T cell reactivity. To identify immunogenic protein component(s) of cell wall protein, T cell lines were established to cell walls and tested against M. leprae proteins separated by SDS-PAGE and transferred to nitrocellulose. Greatest T cell reactivity was observed to proteins of Mr 7 kDa, 16 kDa, and 28 kDa. T cell clones reactive with 7-kDa and 16-kDa Ag from gels failed to respond to proteins of other Mr separated under either reducing or nonreducing conditions, indicating that these molecules are not subunits of larger proteins and may represent monomeric units polymerized into cell walls. The approaches described herein for characterization of immunodominant T cell Ag of M. leprae may be useful for study of T cell Ag in cell walls of bacterial pathogens of man.
Bacterial Proteins↗
Genetic transformation of BCG.
Two substrains of BCG, the Pasteur and Japanese, were successfully transformed with E. coli-mycobacteria shuttle plasmids, constructed from the E. coli plasmid, pIJ666 and the M. fortuitum plasmid, pAL5000. Individual plasmids (pYUB13, pYUB14) were obtained that contain selectable antibiotic resistance markers for kanamycin and chloramphenicol resistance that can replicate in both E. coli and BCG. Transformation of two substrains of BCG was successfully accomplished in 8/14 experiments by means of electroporation, and assessed by the growth of kanamycin-resistant colonies. The E. coli plasmid pIJ666 alone was unable to effect transformation. The results suggest that the M. fortuitum sequences required for transformation function as an origin of replication in BCG. The introduction, persistence and the identity of the plasmids were monitored by re-isolation from consecutive subcultures and restriction analysis. The variables associated with transformation, including the age, viability, and glycine pretreatment of BCG cultures, as well as the electroporation parameters on transformation frequencies are analysed. Consecutive transformations of BCG with plasmid DNA isolated from a BCG transformant increased the efficiency from the level of 10(1)-10(2) obtained with the initial library to 10(3)-10(4) colonies/micrograms DNA with functional pYUB plasmids. The hybrid plasmids were genetically stable and maintained expression of kanamycin resistance in continuous subcultures containing kanamycin for 250 generations. The introduction and stable expression of foreign DNA in BCG on a plasmid vector establishes a basis for the construction of polyvalent recombinant BCG vaccine vehicles expressing not only putative protective mycobacterial antigens, but also antigens for other infectious and malignant diseases.
BCG Vaccine↗
Immunology of bacteria and parasites.
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Animals↗
Microbial glycolipids: possible virulence factors that scavenge oxygen radicals.
Two important pathogens of developing countries, Mycobacterium leprae, the etiologic agent of leprosy, and Leishmania donovani, the protozoal parasite that causes kalaazar, persist in the human host primarily in mononuclear phagocytes. The mechanisms by which they survive in these otherwise highly cytocidal cells are presently unknown. Since the best understood cytocidal mechanism of these cells is the oxygen-dependent system that provides lethal oxidants including the superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH), and singlet oxygen (1O2), we sought specific microbial products of these organisms that might enable them to elude oxidative cytocidal mechanisms. Phenolic glycolipid I of M. leprae and lipophosphoglycan of L. donovani are unique cell-wall-associated glycolipids produced in large amounts by the organisms. In this study, phenolic glycolipid I derivatives and lipophosphoglycan were examined for their ability to scavenge potentially cytocidal oxygen metabolites in vitro. Electron spin resonance and spin-trapping indicate that phenolic glycolipid I derivatives and lipophosphoglycan are highly effective in scavenging hydroxyl radicals and superoxide anions. The results suggest that complex glycolipids and carbohydrates of intracellular pathogens that can scavenge oxygen radicals may contribute to their pathogenicity and virulence.
Animals↗
Regulation of macrophage growth and antiviral activity by interferon-gamma.
Interferons, in addition to their antiviral activity, induce a multiplicity of effects on different cell types. Interferon (IFN)-gamma exerts a unique regulatory effect on cells of the mononuclear phagocyte lineage. To investigate whether the antiviral and antiproliferative effects of IFN-gamma in macrophages can be genetically dissociated, and whether IFN-alpha and IFN-gamma use the same cellular signals and/or effector mechanisms to achieve their biologic effects, we have derived a series of somatic cell genetic variants resistant to the antiproliferative and/or antiviral activities of IFN-gamma. Two different classes of variants were found: those resistant to the antiproliferative and antiviral effects of IFN-gamma against vesicular stomatitis virus (VSV) and those resistant to the antiproliferative effect, but protected against VSV and encephalomyocarditis virus (EMCV) lysis by IFN-gamma. In addition, a third class of mutants was obtained that was susceptible to the growth inhibitory activity, but resistant to the antiviral activity of IFN-gamma. Analysis of these mutants has provided several insights regarding the regulatory mechanisms of IFN-gamma and IFN-alpha on the murine macrophage cell lines. The antiproliferative activity of IFN-gamma on these cells, in contrast to that of IFN-alpha, is mediated by a cAMP-independent pathway. The antiproliferative and antiviral activities of IFN-gamma were genetically dissociated. Variants were obtained that are growth resistant but antivirally protected, or are growth inhibited but not antivirally protected against VSV or EMCV. The genetic analysis indicated that IFN-alpha and IFN-gamma regulate the induction of the dsRNA-dependent P1/eIF-2 alpha protein kinase and 2',5'-oligoadenylate synthetase enzymatic activities via different pathways. Finally, a unique macrophage mutant was obtained that was protected by IFN-gamma against infection by VSV, but not EMCV, suggesting that antiviral mechanisms involved in protection against these different types of RNA viruses must be distinct at some level.
2',5'-Oligoadenylate Synthetase↗
Immunotherapy of localized, intermediate, and diffuse forms of American cutaneous leishmaniasis.
The clinical efficacy of immunotherapy for localized American cutaneous leishmaniasis with a combination of heat-killed Leishmania mexicana amazonensis promastigotes and viable BCG (bacille Calmette Guérin) has been compared with meglumine antimoniate chemotherapy and with BCG alone in a controlled clinical study in 217 patients. The results in the first two groups were comparable, with greater than 90% clinical cures with an average time of 16-18 w required for healing. The cure rate was considerably lower (42%) and more prolonged in the group receiving BCG alone. Secondary effects were observed in less than 5% of the patients receiving combined immunotherapy or BCG alone. In contrast, 49% of the patients receiving chemotherapy showed side effects. High therapeutic efficacy was also observed using combined immunotherapy in patients with intermediate and diffuse cutaneous leishmaniasis who were previously unresponsive to chemotherapy. Cure or clinical improvement was seen in all 11 patients with intermediate forms of the disease, and marked clinical improvement was observed in 9 of 10 patients with diffuse disease. The results on the efficacy of the combined vaccine in immunotherapy for American cutaneous leishmaniasis provide a strong rationale for studying its effectiveness in prophylactic trials.
Adolescent↗
On the mechanism of human T cell suppression.
Previous evidence from several laboratories suggests that CD8+ T suppressor cells may be important regulatory elements governing specific unresponsiveness of lepromatous lepromatous leprosy patients to M.leprae. To analyse the mechanism of suppression, CD8+ Ts clones were established from lesions and peripheral blood of lepromatous patients and tested for ability to suppress antigen-responsive CD4+. Th clones or PBL. Suppression required induction by specific M.leprae antigen, but was effected in an antigen-non-specific fashion. The Ts clones failed to exhibit cytotoxicity of four antigen-exposed MHC-matched target cells: (i) an ori-SV40 transformed macrophage line; (ii) EBV transformed B cell lines; (iii) primary macrophages; and (iv) M.leprae responsive CD4+ cells. The possibility that Ts clones induce functional inactivation of CD4+ clones in vitro was investigated. M.leprae-responsive CD4+ clones were preincubated with Ts CD8+ clones, APC, and antigen for 16 h, after which the CD8+ cells were removed. The CD4+ clones with M.leprae and APC remained unresponsive to restimulation with APC and antigen for at least 10 days, although they responded to IL-2. Addition of IL-2 to the pre- or post-incubation cultures neither prevented the induction of unresponsiveness, nor reversed it. Earlier models of tolerance have suggested that receptor occupancy in the absence of second signals induces tolerance in B and T cells. Under conditions in which antigen responses of Th clones were HLA-DR-restricted, the Ts clones were able to suppress the response of DR mismatched Th clones. Thus, the effect of the Ts cells, like mechanisms requiring antigen presentation without a second signal, appears to be induction of clonal anergy in Th cells, perhaps by a novel mechanism.
Antigen-Presenting Cells↗
New strategies for leprosy and tuberculosis and for development of bacillus Calmette-Guérin into a multivaccine vehicle.
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Antigens, Bacterial↗
Genetic relationships among Mycobacterium leprae, Mycobacterium tuberculosis, and candidate leprosy vaccine strains determined by DNA hybridization: identification of an M. leprae-specific repetitive sequence.
Comparative DNA hybridization studies of genomic DNA indicated that, while different isolates of armadillo-derived Mycobacterium leprae have a high degree of homology, binding of M. leprae genomic DNA to DNA of other species of mycobacteria or to corynebacteria was low, establishing that M. leprae is only remotely genetically related to any of the species examined. Several candidate leprosy vaccine mycobacterial strains were similarly found to have little genetic similarity to M. leprae. In contrast, the DNAs of the slow-growing mycobacteria M. tuberculosis, M. africanum, M. bovis, and M. microti were found to be very closely related. In the course of these studies, an M. leprae-specific repetitive sequence, greater than 15-fold per genome equivalent, was identified that might be useful for diagnostic and epidemiological studies.
Antigens, Bacterial↗
Molecular cloning of a gene selectively induced by gamma interferon from human macrophage cell line U937.
A cDNA clone encoding a gamma interferon (IFN-gamma)-inducible mRNA in human cells of the macrophage lineage was isolated and characterized. The corresponding gene, gamma.1, was selectively induced by IFN-gamma, responding a hundredfold better to IFN-gamma than to IFN-alpha. The induction was rapid and transient, with maximal mRNA accumulation at about 3 h and decline to the basal level after 48 h. Transcriptional activation could be detected as early as 5 min after IFN-gamma stimulation and accounted entirely for the mRNA accumulation. The induction of gamma.1 by IFN-gamma was cell-type restricted, being seen only in macrophages and endothelial cells. In addition, phorbol ester-induced differentiation of promyelocytic HL-60 cells and promonocytic THP-1 cells rendered the gamma.1 gene inducible by IFN-gamma. The 1.0-kilobase gamma.1 cDNA sequence encoded a small predicted polypeptide of 38 amino acids and had a conserved sequence associated with rapidly turning over mRNAs. In vitro translation of the gamma.1 transcript yielded a 4,000-dalton polypeptide.
Amino Acid Sequence↗
Development of genetic systems for the mycobacteria.
Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system which allows for the transfer, mutation, and expression of specific genes. Genetic analysis of mycobacteria has been exceedingly difficult since the mycobacteria grow slowly and no natural efficient method of gene transfer within the pathogenic has thus far been found. Using a molecular genetic approach, we have developed both the vectors and the methodology for efficient gene transfer in the mycobacteria. Initially, a novel of type of mycobacteriophage vector was developed, termed a shuttle phasmid. This hybrid shuttle vector replicates in Escherichia coli as a plasmid and in mycobacteria as a phage, capable of introducing foreign DNA into a wide variety of mycobacterial species. A set of shuttle phasmids, constructed from a temperate mycobacteriophage, retained their ability to lysogenize their mycobacterial hosts and could thus introduce foreign DNA stably into mycobacterial cells. An E. coli gene conferring kanamycin-resistance was cloned into these vectors and shown to express in the mycobacteria, thus providing the first selectable marker gene for subsequent genetic studies. Using kanamycin-resistance gene as a selection, the M. fortuitum plasmid pAL5000 replicon, and electroporation; a plasmid transformation system has been developed for both M. smegmatis and BCG. We now plan to use these phage and plasmid systems to analyze, genetically, the virulence attributes of the pathogenic mycobacteria. In addition, by introducing and expressing foreign antigens in BCG, we hope to develop a novel recombinant multi-vaccine vehicle capable of conferring immunity to a variety of bacterial, viral, and parasitic pathogens.
BCG Vaccine↗
Transformation of BCG with plasmid DNA.
Pasteur 1173P2 and Japanese 172 BCG substrains were transformed with plasmid pAL5000::plJ666 by electroporation and assessed by the growth of kanamycin-resistant colonies. From the transformants pYUP plasmids were isolated containing plJ666 inserted into pAL5000. The introduction, persistence and the identity of the plasmid were monitored by reisolation from consecutive subcultures and restriction analysis. The effect of variables--age, viability, glycine pretreatment of BCG cultures, electroporation parameters--on transformation frequencies were analyzed. Consecutive transformation of BCG with plasmid DNA isolated from a BCG transformant increased the efficiency from the level of 10(1)-10(2) obtained with the initial library to 10(3)-10(4) colonies/micrograms DNA with the pYUB plasmids. Maintenance of plasmid and expression of kanamycin resistance in continuous subcultures were stable for 100 generations from the first successful transformation to the present. The introduction and stable expression of foreign DNA in BCG on a plasmid vector establishes a basis for the construction of polyvalent recombinant BCG vaccine vehicles expressing not only putative protective mycobacterial antigens, but also antigens for other infectious and malignant diseases.
BCG Vaccine↗
An ordinary mortal's guide to the molecular biology of mycobacteria.
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DNA Replication↗
Vaccine development. On relating immunology to the Third World: some studies on leprosy.
Leprosy is of interest to immunologists because the varied clinical manifestations of the disease correlate closely with the immunological spectrum. Resistance to infection is dependent on appropriate cell-mediated immunity, but patients with the lepromatous form fail to respond to antigens of M. leprae. In vitro studies have revealed the existence of T-suppressor cells of the phenotype CD8+, CD3+, HLA-DR+, FcR+, 9.3-, which are restricted by major histocompatibility complex (MHC) class II antigens. Several new candidate vaccines against leprosy have been effective in breaking immunological unresponsiveness and engendering cell-mediated immunity in lepromatous leprosy patients, including the combination of BCG+ killed M. leprae. Because BCG has unique adjuvant properties, we have begun to use molecular genetic approaches to develop BCG into a multivaccine vehicle capable of immunizing simultaneously against several pathogens. Both phage-based and plasmid-based strategies have been successfully developed for introducing selectable markers into BCG for the first time.
Bacterial Vaccines↗