Characterization of a 10- to 14-kilodalton protease-sensitive mycobacterium tuberculosis H37Ra antigen that stimulates human -yi T cells

gamma delta T-cell receptor-bearing T cells (gamma delta T cells) are readily activated by intracellular bacterial pathogens such as Mycobacterium tuberculosis. The bacterial antigens responsible for gamma delta T-cell activation remain poorly characterized. We have found that heat treatment of live M. tuberculosis bacilli released into the supernatant an antigen which stimulated human gamma delta T cells, gamma delta T-cell activation was measured by determining the increase in percentage of gamma delta T cells by flow cytometry in peripheral blood mononuclear cells stimulated with antigen and by proliferation of gamma delta T-cell lines with monocytes as antigen-presenting cells. Supernatant from heat-treated M. tuberculosis was fractionated by fast-performance liquid chromatography (FPLC) on a Superose 12 column. Maximal gamma delta T-cell activation was measured for a fraction of 10 to 14 kDa. Separation of the supernatant by preparative isoelectric focusing demonstrated peak activity at a pi of <4.0. On two-dimensional gel electrophoresis, the 10- to 14-kDa FPLC fraction contained at least seven distinct molecules, of which two had a pi of <4.5. Protease treatment reduced the bioactivity of the 10- to 14-kDa FPLC fraction for both resting and activated gamma delta T cells. Murine antibodies raised to the 10- to 14-kDa fraction reacted by enzyme-linked immunosorbent assay with antigens of 10 to 14 kDa in lysate of M. tuberculosis. In addition, gamma delta T cells proliferated in response to an antigen of 10 to 14 kDa present in M. tuberculosis lysate. gamma delta T-cell-stimulating antigen was not found in culture filtrate of M. tuberculosis but was associated,vith the bacterial pellet and lysate of M. tuberculosis. These results provide a preliminary characterization of a 10- to 14-kDa, cell-associated, heat-stable, low-pI protein antigen of M. tuberculosis which is a major stimulus for human gamma delta T cells.

Cytotoxic T lymphocytes raised against Japanese encephalitis virus: effector cell phenotype, target specificity and in vitro virus clearance.

Several H-2 defined cell lines were examined for their ability to support infection and replication of Japanese encephalitis virus (JEV) before their use in in vitro and in vivo stimulation protocols for generating cytotoxic T lymphocytes (CTLs) against JEV. Among II different cell lines tested, two H-2(d) macrophage tumour lines (P388D1, RAW 264.7), an H-2(d) hybridoma (Sp2/0), an H-2K(k)D(d) neuroblastoma (Neuro 2a), and H-2(k) fibroblast cell line (L929) were found to support JEV infection and replication. These cell lines were used to generate anti-JEV CTLs by using in vivo immunization followed by in vitro stimulation of BALB/c mice. We observed that not only syngeneic and allogeneic infected cells but also JEV-infected xenogeneic cells could prime BALB/c mice for the generation of JEV-specific CTLs upon subsequent in vitro stimulation of splenocytes with JEV-infected syngeneic cells. Although infected xenogeneic cells were used for immunization, the anti-JEV effecters that were generated lysed infected syngeneic targets but not JEV-infected xenogeneic or allogeneic target cells in a 5h Cr-51 release assay. These anti-JEV effecters recognized syngeneic target cells infected with West Nile virus to a lesser extent and were shown to be Lyt-2.2(+) T cells. The results of unlabelled cold target competition studies suggested alterations in the cell surface expression of viral antigenic determinants recognized by these CTLs. We further demonstrate that the JEV-specific CTLs generated could virtually block the release of infectious virus particles from infected P388D1 and Neuro 2a cells in vitro.

Inhibition of nuclear protein import by a monoclonal antibody against a novel class of nuclear pore proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab Ea-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS-albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small nonnuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex. (C) 1994 Academic Press, Inc.

Recombinant mid gut antigen (Bm95) as a vaccine against Indian Rhiphicephalus haemaphysaloides in Bos indicus cattle

In the present study, we report for the first time the efficacy of recombinant Bm95 mid gut antigen isolated from an Argentinean strain of Rhipicephalus microplus strain A in controlling the tick infestations in India. The synthetic gene for Bm95 optimized for expression in yeast was obtained and used to generate yeast transformants expressing Bm95 which was purified to apparent homogeneity. Liquid chromatography-mass spectrometry analysis of the purified protein confirmed its identity as Bm95. Vaccine was prepared by blending various concentrations of purified Bm95 with aluminium hydroxide as an adjuvant. lmmunogenicity studies of the vaccine in rabbits and cattle indicated that the vaccine was highly immunogenic. The efficacy studies of the vaccine was done in cattle. Naive Bos indicus cattle were vaccinated with the recombinant vaccine and were challenged with the larval, nymphal and adult forms of Rhiphicephalus haemaphysaloides. The vaccine protected the animals from larval, nymph and adult tick challenges with an efficacy of 98.7%, 84.6% and 78.9% respectively. The results obtained from the above studies clearly demonstrated the advantage and possibilities of the use of Bm95 in controlling R. haemaphysaloides infestations in the field. (C) 2010 Elsevier Ltd. All rights reserved.

Immune responses to hemagglutinin-neuraminidase protein of peste des petits ruminants virus expressed in transgenic peanut plants in sheep

Peste des petits ruminants (PPR) is an acute, highly contagious disease of small ruminants caused by a morbillivirus, Peste des petits ruminants virus (PPRV). The disease is prevalent in equatorial Africa, the Middle East, and the Indian subcontinent. A live attenuated vaccine is in use in some of the countries and has been shown to provide protection for at least three years against PPR. However, the live attenuated vaccine is not robust in terms of thermotolerance. As a step towards development of a heat stable subunit vaccine, we have expressed a hemagglutinin-neuraminidase (HN) protein of PPRV in peanut plants (Arachis hypogea) in a biologically active form, possessing neuraminidase activity. Importantly. HN protein expressed in peanut plants retained its immunodominant epitopes in their natural conformation. The immunogenicity of the plant derived HN protein was analyzed in sheep upon oral immunization. Virus neutralizing antibody responses were elicited upon oral immunization of sheep in the absence of any mucosal adjuvant. In addition, anti-PPRV-HN specific cell-mediated immune responses were also detected in mucosally immunized sheep. (C) 2010 Elsevier B.V. All rights reserved.

Dissociation of monoclonal antibody-antigen complexes: Implications for ELISA procedures

A single step solid phase radioimmunoassay (SS-SPRIA) has been developed for human chorionic,gonadotropin (hCG) using monoclonal antibodies (MAb) from culture media adsorbed immunochemically on plastic tubes. The assays have been found to be very simple in terms of operation and do not demand purification of MAbs. Several MAbs which do not show any displacement in liquid phase RIA and ELISA provide a satisfactory SS-SPRIA. Our investigations revealed that the assumption regarding the stability of the primary Mab-Ag complex during incubation and washing steps in ELISAs is not strictly valid for dissociable MAbs. A comparison of different assay systems suggests that the single step SPRIA offers additional advantages over conventionally used multistep ELISA procedures and provides a quantitative probe for the analysis of epitope-paratope interactions.

Characterization of an in vitro transcription system from rinderpest virus

An in vitro transcription system for rinderpest virus (RPV) is described. Ribonucleoprotein complexes isolated from RPV-infected Vero cells, human lung carcinoma cells, or detergent-disrupted purified virions synthesized authentic RPV mRNAs for the N, P, M, F and H genes as identified by dot blot hybridization analysis with individual cDNA clones. The relative abundance of the mRNAs synthesized in vitro decreased from the 3? end of the genome to the 5? end, very similar to that observed with measles virus transcription in vitro. The transcription by purified virions was stimulated three-fold by the addition of infected human lung carcinoma cell lysate, demonstrating the involvement of host factor(s) in mRNA synthesis.

Molecular and serological studies on the recent seal virus epizootics in Europe and Siberia

The virus epizootics which occurred in seals in both Europe and Siberia during 1987/1988 were caused by two different morbillivirus, referred to as phocid distemper virus (PDV) 1 and 2, respectively. Molecular and serological studies have shown that the European virus is quite distinct from canine distemper virus (CDV), its closest relative in the morbillivirus group. Analysis of tissues obtained from infected seals from a wide geographical distrubution over Northern Europe showed that the infectious agent (PDV 1) was identical in all cases. Nucleotide sequence analysis of one of the virus genes suggested that this virus has evolved away from CDV over a long time period and is most probably an enzootic virus of marine mammals. In contrast, the virus (PDV 2) which caused the deaths of many Siberian seals was indistinguishable, both serologically and at the molecular level, from CDV and must have originated from a land source.

Interactions of 3' terminal and 5' terminal regions of physalis mottle virus genomic RNA with its replication complex

Physalis mottle virus (PhMV) belongs to the tymogroup of positive-strand RNA viruses with a genome size of 6 kb. Crude membrane preparations from PhMV-infected Nicotiana glutinosa plants catalyzed the synthesis of PhMV genomic RNA from endogenously bound template. Addition of exogenous genomic RNA enhanced the synthesis which was specifically inhibited by the addition of sense and antisense transcripts corresponding to 3' terminal 242 nucleotides as well as the 5' terminal 458 nucleotides of PhMV genomic RNA while yeast tRNA or ribosomal RNA failed to inhibit the synthesis. This specific inhibition suggested that the 5' and 3' non-coding regions of PhMV RNA might play an important role in viral replication.

Protection of adult but not newborn mice against lethal intracerebral challenge with Japanese encephalitis virus by adoptively transferred virus-specific cytotoxic T lymphocytes: Requirement for L3T4(+) T cells

The protective ability of cytotoxic T cells (CTL) raised in vitro against Japanese encephalitis virus (JEV) was examined by adoptive transfer experiments. Adoptive transfer of anti-JEV effecters by intracerebral (i.c.) but not by intraperitoneal (i.p.) or intravenous (i.v.) routes protected adult BALB/c mice against lethal i.c. JEV challenge. In contrast to adult mice, adoptive transfer of anti-JEV effecters into newborn (4-day-old) and suckling (8-14-day-old) mice did not confer protection. However, virus-induced death was delayed in suckling mice compared to newborn mice upon adoptive transfer. The specific reasons for lack of protection in newborn mice are not clear but virus load was found to be higher in newborn mice brains compared to those of adults and virus clearance was observed only in adult mice brains but not in newborn mice brains upon adoptive transfer. Specific depletion of Lyt 2.2(+), L3T4(+) or Thy-1(+) T cell populations before adoptive transfer abrogated the protective ability of transferred effecters. However, when Lyt 2.2(+) cell-depleted and L3T4(+) cell-depleted effecters were mixed and transferred into adult mice the protective activity was retained, demonstrating that both Lyt 2.2(+) and L3T4(+) T cells are necessary to confer protection. Although the presence of L3T4(+) T cells in adoptively transferred effector populations enhanced virus-specific serum neutralizing antibodies, the presence of neutralizing antibodies alone without Lyt 2.2(+) cells was not sufficient to confer protection.

Realtime kinetic analysis of antigen-antibody interaction using solid phase binding: Transformation of hCG-monoclonal antibody complex

Kinetic constants of MAb-hCG interactions have been determined using solid phase binding of I-125[hCG] to immobilized MAb. While association has been shown to follow the expected pattern, dissociation consists of at least two reversible steps, one with a rate constant of 0.0025 min(-1), and a second with a rate constant of 0.00023 min(-1). Validity of affinity constant measurements in the light of the complex reaction kinetics is discussed, A comparison between the method of surface plasmon resonance technology (BIAcore) and solid phase binding (SPB) for determination of kinetic parameters shows that SPB provides not only a cost-effective approach for determination of realtime kinetic parameters of macromolecular ligand-ligate interaction but also a method with several advantages over the BIAcore system in investigating the mechanism of antigen-antibody interaction.