Evidence for N7 guanine methyl transferase activity encoded within the modular domain of RNA-dependent RNA polymerase L of a Morbillivirus.

Post-transcriptional modification of viral mRNA is essential for the translation of viral proteins by cellular translation machinery. Due to the cytoplasmic replication of Paramyxoviruses, the viral-encoded RNA-dependent RNA polymerase (RdRP) is thought to possess all activities required for mRNA capping and methylation. In the present work, using partially purified recombinant RNA polymerase complex of rinderpest virus expressed in insect cells, we demonstrate the in vitro methylation of capped mRNA. Further, we show that a recombinant C-terminal fragment (1717-2183 aa) of L protein is capable of methylating capped mRNA, suggesting that the various post-transcriptional activities of the L protein are located in independently folding domains.

Molecular markers for discriminating Streptococcus pyogenes and S. dysgalactiae subspecies equisimilis.

Given the increasing aetiological importance of Streptococcus dysgalactiae subspecies equisimilis in diseases which are primarily attributed to S. pyogenes, molecular markers are essential to distinguish these species and delineate their epidemiology more precisely. Many clinical microbiology laboratories rely on agglutination reactivity and biochemical tests to distinguish them. These methods have limitations which are particularly exacerbated when isolates with mixed properties are encountered. In order to provide additional distinguishing parameters that could be used to unequivocally discriminate these two common pathogens, we assess here three molecular targets: the speB gene, intergenic region upstream of the scpG gene (IRSG) and virPCR. Of these, the former two respectively gave positive and negative results for S. pyogenes, and negative and positive results for S. dysgalactiae subsp. equisimilis. Thus, a concerted use of these nucleic acid-based methods is particularly helpful in epidemiological surveillance to accurately assess the relative contribution of these species to streptococcal infections and diseases.

Host factor Ebp1 inhibits rinderpest virus transcription in vivo.

ErbB3 binding protein Ebp1 has been shown to downregulate ErbB3 receptor-mediated signaling to inhibit cell proliferation. Rinderpest virus belongs to the family Paramyxoviridae and is characterized by the presence of a non-segmented negative-sense RNA genome. In this work, we show that rinderpest virus infection of Vero cells leads to the down-regulation of the host factor Ebp1, at both the mRNA and protein levels. Ebp1 protein has been shown to co-localize with viral inclusion bodies in infected cells, and it is packaged into virions, presumably through its interaction with the N protein or the N-RNA itself. Overexpression of Ebp1 inhibits viral transcription and multiplication in infected cells, suggesting that a mutual antagonism operates between host factor Ebp1 and the virus.

Detection of interferon gamma-secreting CD8+ T lymphocytes in humans specific for three PE/PPE proteins of Mycobacterium tuberculosis.

The PE and PPE family of proteins of Mycobacterium tuberculosis have been hypothesized to play important roles in the biology of the organism and some proteins have been shown to be involved in eliciting T-cell responses. Earlier, we had identified putative HLA class I binding epitopes of the PE and PPE proteins of Mycobacterium tuberculosis employing computational and molecular modeling approaches. In the present work, three of the PE/PPE family proteins, coded by Rv1818c, Rv3812 and Rv3018c genes, were selected based on the computational analysis for testing human immune responses. PBMCs from patients with active tuberculosis and healthy, BCG vaccinated, PPD-positive individuals were tested for in vitro proliferative response and gamma-interferon production using synthetic peptides derived from the chosen proteins. Significant differences were seen in the responsiveness between healthy controls and patients. Antigen-specific T-cell lines were established from the PBMCs of healthy controls and their responses to peptide-specific CD8+ T-cell effectors were shown to be present at high frequency in the PBMCs of PPD+ controls. The T-cell lines also showed cytotoxic activity against the peptide pulsed monocytes.

Recombinant L and P protein complex of Rinderpest virus catalyses mRNA synthesis in vitro.

Rinderpest virus belongs to the family of Paramyxoviridae, consisting of non-segmented negative sense RNA viruses. Viral transcription and replication are carried out by the RNA dependent RNA polymerase L protein which functions together with P protein as L-P complex. The exact events triggering the polymerase complex from transcription to replication function is poorly understood. In the present work, an in vitro transcription system has been described with partially purified L-P complex expressed in insect cells and viral genomic RNA. The relative abundance of each species of mRNA synthesized in vitro decreased from the 3' end of the genome to the 5' end similar to their abundance in virus infected cells. Recombinant L-P complex was unable to synthesize leader RNA suggesting the initiation of transcription from gene start site and not at the 3' end of the genome.

HLA-A*0201-restricted cytotoxic T-cell epitopes in three PE/PPE family proteins of Mycobacterium tuberculosis.

CD8+ T cells are thought to play an important role in protective immunity against tuberculosis. We report the identification of three peptides derived from Rv1818c, Rv3812 and Rv3018c proteins of Mycobacterium tuberculosis that bound to HLA-A*0201 molecules and their ability to induce in vitro T-cell response in peripheral blood lymphocytes from HLA-A*0201-positive healthy individuals (PPD+) and patients with TB. The peptide-specific cytotoxic T lymphocytes (CTL) generated were capable of recognizing peptide pulsed targets. Three 9-mer peptides bound with high affinity to HLA-A*0201 and displayed low dissociation rates of the bound peptide from HLA. Epitope-specific recognition was demonstrated by the release of perforin and gamma-interferon. Overall, our results demonstrate the presence of HLA class I-restricted CD8+ CTL against proteins from PE and PPE proteins of M. tuberculosis and identify epitopes that are strongly recognized by HLA-A*0201-restricted CD8+ T cells in humans. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.

Characterization of T-cell immunogenicity of two PE/PPE proteins of Mycobacterium tuberculosis.

The PE and PPE proteins of Mycobacterium tuberculosis form a source of antigenic variation among different strains of this bacterium. Two of the PE_PGRS protein-encoding genes, rv3812 and rv3018c, are expressed in pathogenic mycobacteria and are implicated, respectively, in the persistence of the organism in macrophages and in virulence. Peptides derived from these proteins have been predicted to bind major histocompatibility complex (MHC) class I with high affinity on the basis of immunoinformatics analysis, suggesting a possible role for these proteins in antimycobacterial immunity. In the present work, using DNA constructs containing the rv3812 and rv3018c genes of M. tuberculosis, the immunogenicity of these proteins was demonstrated in BALB/c mice. Immunization with either DNA construct induced a significant number of CD8+-type T cells and a strong Th1-type response, with high gamma interferon (IFN-gamma) and low interleukin-4 responses. Three nonameric peptides of Rv3812 and two of Rv3018c elicited a strong T-cell response in an MHC-restricted manner. An epitope-specific response was demonstrated by the lysis of peptide-pulsed antigen-presenting cells, release of perforin and IFN-gamma production. Experimentally, these peptides bound with high affinity to MHC H-2Kd and showed low dissociation rates of peptide-MHC complexes. This study suggests that the identified T-cell epitopes may contribute to immunity against tuberculosis if included in a vaccine.

Detection of peste des petits ruminants virus antigen using immunofiltration and antigen-competition ELISA methods.

Peste des petits ruminants (PPR) is one of the most economically important diseases affecting sheep and goats in India. An immunofiltration-based test has been developed using either mono-specific serum/monoclonal antibodies (mAb) prepared against a recombinant truncated nucleocapsid protein of rinderpest virus (RPV) cross-reactive with PPR virus. This method consists of coating ocular swab eluate from suspected animals onto a nitrocellulose membrane housed in a plastic module, which is allowed to react with suitable dilutions of a mAb or a mono-specific polyclonal antibody. The antigen-antibody complex formed on the membrane is then detected by protein A-colloidal gold conjugate, which forms a pink colour. In the immunofiltration test, concordant results were obtained using either PPRV mAb or mono-specific serum. Another test, an antigen-competition ELISA which relies on the competition between plate-coated recombinant truncated 'N' protein of RPV and the PPRV 'N' protein present in ocular swab eluates (sample) for binding to the mono-specific antibody against N protein of RPV (in liquid phase) was developed. The cut-off value for this test was established using reverse transcription polymerase chain reaction (RT-PCR) positive and negative oculo-nasal swab samples. Linear correlation between percent inhibition (PI) values in antigen-competition ELISA and virus infectivity titres was 0.992. Comparison of the immunofiltration test with the antigen-competition ELISA yielded a sensitivity of 80% and specificity of 100%. These two tests can serve as a screening (immunofiltration) and confirmatory (antigen-competition ELISA) test, respectively, in the diagnosis of PPR in sheep or goats.

Peptidomimics of antigen are present in variable region of heavy and light chains of anti-idiotypic antibody and function as surrogate antigen for perpetuation of immunological memory.

Peptide antigens composed of relevant B cell and T cell epitopes, capable of inducing protective immune response against the whole pathogen, are potentially safe, alternative vaccine antigens for prevention of wide range of diseases. Here, we show that short peptides derived from internal image sequences of anti-idiotypic antibody (peptidomimics) can function as both B and T cell epitopes and perpetuate antigen specific immunological memory. We have sequenced the variable regions of heavy and light chains of the anti-idiotypic antibody specific to rinderpest virus hemagglutinin protein and predicted T cell epitopes in these sequences by an immuno-informatics approach. We have studied the interaction of these epitopes with MHC class I by in vitro assays and in silico analysis by molecular modeling of the idiopeptide-MHC complexes as well as antigen-derived peptide-MHC complexes. The functional capacity of anti-idiotypic antibody derived peptides to stimulate antigen specific T cells in vitro was tested. The ability of peptidomimics to proliferate the immune splenocytes in vitro was 10 times more when compared with that of a control peptide taken from the constant region of immunoglobulin. Similarly three- to fivefold more amounts of IL-2 and IFN-gamma were secreted by immune splenocytes in response to in vitro re-stimulation with peptidomimics. Further, we have provided evidence for the generation of antibodies against peptidomimics in memory response generated on antigen or anti-idiotypic antibody immunizations. In summary, our experiments suggest that peptidomimics are generated in the body after antigen immunization and may have important roles in vivo in regulating antigen specific immunological memory.

Perpetuation of immunological memory through common MHC-I binding modes of peptidomimic and antigenic peptides.

Understanding the molecular mechanisms of immunological memory assumes importance in vaccine design. We had earlier hypothesized a mechanism for the maintenance of immunological memory through the operation of a network of idiotypic and anti-idiotypic antibodies (Ab2). Peptides derived from an internal image carrying anti-idiotypic antibody are hypothesized to facilitate the perpetuation of antigen specific T cell memory through similarity in peptide-MHC binding as that of the antigenic peptide. In the present work, the existence of such peptidomimics of the antigen in the Ab2 variable region and their similarity of MHC-I binding was examined by bioinformatics approaches. The analysis employing three known viral antigens and one tumor-associated antigen shows that peptidomimics from Ab2 variable regions have structurally similar MHC-I binding patterns as compared to antigenic peptides, indicating a structural basis for memory perpetuation.

Evaluation of T-cell responses to peptides with MHC class I-binding motifs derived from PE_PGRS 33 protein of Mycobacterium tuberculosis.

The PE and PPE proteins of Mycobacterium tuberculosis form a source of antigenic variation among different strains of M. tuberculosis. One of the PE_PGRS proteins, Rv1818c, plays a role in the pathogenesis of mycobacterial infection and specifically influences host-cell responses to tuberculosis infection. Although little is known about these two classes of protein, an immunoinformatics approach has indicated the possibility of their participation in eliciting a major histocompatibility complex (MHC) class I-mediated immune response against tuberculosis, as peptides derived from Rv1818c are predicted to bind to MHC class I molecules with high affinity. In the present work, a DNA vaccine was constructed encoding the full-length Rv1818c protein of M. tuberculosis and its immunogenicity was analysed in BALB/c mice. Immunization with Rv1818c DNA induced a strong CD8+ cytotoxic lymphocyte and Th1-type response, with high levels of gamma interferon (IFN-gamma) and low levels of interleukin-4. Two nonameric peptides (Peptide(6-14) and Peptide(385-393)) from Rv1818c were identified by their ability to induce the production of IFN-gamma by CD8+ T cells in mice immunized with Rv1818c DNA. An epitope-specific response was demonstrated by the lysis of peptide-pulsed antigen-presenting cells, release of cytotoxic granules and IFN-gamma production. These peptides bound with high affinity to MHC H-2K(d) and showed low dissociation rates of peptide-MHC complexes. These results could form the basis for testing the identified T-cell epitopes of PE_PGRS proteins in the induction of protective immunity against M. tuberculosis challenge in the mouse model.

Recombinant idiotypic TCRbeta chain immunization in mice generates antigen specific T cell response.

Vaccination remains the most cost-effective means of preventing infectious diseases. Success of vaccination depends on generation of effective memory response. Understanding the mechanism of generation and maintenance of immunological memory would help in the design of rational vaccines. T lymphocytes play a central role in the generation of protective immune response against many microbial infections. A hypothesis known as relay hypothesis was earlier proposed, which explains the maintenance of immunological memory through interaction of idiotypic and anti-idiotypic lymphocytes. In the present study, we have shown that immunization with a model antigen, chicken ovalbumin specific T cell receptor beta chain (idiotypic TCR) generates TCR specific antibody and anti-idiotypic T cell responses as well as ovalbumin specific T cell response. We further show that boosting of ovalbumin primed mice with ovalbumin specific idiotypic TCRbeta DNA or TCRbeta protein gives memory response for ovalbumin. This study provides experimental evidence for perpetuation of immunological memory through idiotypic network interactions.

A combined immuno-informatics and structure-based modeling approach for prediction of T cell epitopes of secretory proteins of Mycobacterium tuberculosis.

The role of secretory proteins of Mycobacterium tuberculosis in pathogenesis and stimulation of specific host responses is well documented. They are also shown to activate different cell types, which subsequently present mycobacterial antigens to T cells. Therefore identification of T cell epitopes from this set of proteins may serve to define candidate antigens with vaccine potential. Fifty-two secretory proteins of M. tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nonameric peptides. All possible overlapping nonameric peptide sequences from 52 secretory proteins were generated in silico and analyzed for their ability to bind to 33 alleles belonging to A, B and C loci of HLA class I. Fifteen percent of generated peptides are predicted to bind to HLA with halftime of dissociation T(1/2) >or=100 min and 73% of the peptides predicted to bind are mono-allelic in their binding. The structural basis for recognition of no-namers by different HLA molecules was studied employing structural modeling of HLA class I-peptide complexes and there exists a good correlation between structural analysis and binding prediction. Pathogen peptides that could behave as self- or partially self-peptides in the host were eliminated using a comparative study with the human proteome, thus reducing the number of peptides for analysis. The implications of the finding for vaccine development are discussed vis-à-vis the limitations of the use of subunit vaccine and DNA vaccine.

Idiotypic T cells specific for Morbillivirus nucleocapsid protein process and present their TCR to cognate anti-idiotypic CD8+ T cells.

CD8(+) T cells are activated by the presentation of antigenic peptide through MHC class I molecules. Newly synthesized proteins formed as defective ribosomal products (DRiPs) can act as a major source of antigenic peptides for MHC class I presentation pathway. Majority of these peptides are generated from the intracellular degradation of self antigens. In the present study, we have shown that newly synthesized T cell receptor (TCR) beta chains formed as DRiPs in T cells are ubiquitinated and degraded by the proteasomes. These TCR-DRiPs are processed and presented by activated T cells to cognate anti-idiotypic CD8(+) T cells. Presentation of TCR idiopeptide (peptide derived from the variable region of idiotypic TCR) by activated T cells leads to Bcl-2 expression and cytokine secretion by anti-idiotypic CD8(+) T cells. Presentation of intracellular antigen by T cells may have important implications in immunoregulation, control of lymphotropic virus infection and autoimmune diseases.

Perpetuation of immunological memory: role of serum antibodies and accessory cells.

A self-sustaining mechanism for perpetuation of immunological memory is proposed which requires the involvement of idiotypic-anti-idiotypic antibody immune-complexes, antigen-presenting cells and follicular dendritic cells, but not long-lived memory cells or persisting antigens.

Diagnostic Disparity in Solitary Cysticercosis of the Forearm in a Child.

Solitary cysticercosis of muscle is a rare disease causing diagnostic dilemma. Cysticercosis commonly affects the central nervous system and other tissues by dissemination imposing a serious health problem. We report this rare presentation of solitary cysticercosis of flexor digitorum superficialis in a five year old otherwise healthy child. The fine needle aspiration cytology and histopathological diagnosis were inconclusive but ultrasonography of the muscle clinched the diagnosis.

The hemagglutinin-neuraminidase protein of peste des petits ruminants virus is biologically active when transiently expressed in mammalian cells.

The genes coding for the surface glycoproteins hemagglutinin-neuraminidase (HN) of the peste des petits ruminants virus (PPRV) and hemagglutinin (H) of rinderpest virus (RPV) were cloned in a cytomagalovirus promoter driven expression vector and expressed transiently in mammalian cells. The protein expression was apparent 24 h after transfection and the expressed proteins were detected at the cell surface. The transiently expressed PPRV HN protein was found to be biologically active in possessing hemadsorption and neuraminidase activities. On the other hand, RPV H protein exhibited neuraminidase activity but was deficient in hemadsorption activity. The substrate specificity of the neuraminidase activity of these two proteins differed distinctly. The presence of neuraminidase activity in both PPRV HN and RPV H proteins is unusual among members of the morbillivirus genus.

Development of a reconstitution system for Rinderpest virus RNA synthesis in vitro.

The RNA dependent RNA polymerase of Rinderpest virus consists of two subunits-the large protein (L) and the phosphoprotein (P), where L is thought to be responsible for the catalytic activities in association with P protein which plays multiple roles in transcription and replication. The nucleocapsid protein (N) is necessary for encapsidation of genomic RNA, which is required as N-P complex. To understand the different steps of transcription and replication as well as the roles played by the three proteins, an in vitro reconstitution system for RNA synthesis is necessary which is not available for any morbillivirus. We describe here, an in vitro reconstitution system for transcription and replication of Rinderpest virus utilizing a synthetic, positive sense N-RNA minigenome template, free of endogenous viral polymerase proteins and recombinant viral proteins (P+L and P+N) expressed in insect cells by recombinant baculoviruses. We show that although L-P complex is sufficient to synthesize negative sense minigenome RNA, soluble N protein is necessary for encapsidation of RNA as well as synthesis of (+) sense leader RNA and (+) sense minigenome RNA.

Effect of single amino acid mutations in the conserved GDNQ motif of L protein of Rinderpest virus on RNA synthesis in vitro and in vivo.

The paramyxovirus RNA-dependent RNA polymerase consists of two subunits, the transcription co-factor phosphoprotein P and the large protein L, which possesses all the catalytic functions such as RNA synthesis (both transcription replication), methylation, capping and polyadenylation. The L protein has high sequence homology among the negative sense RNA viruses. The domains and residues on the L protein involved in the above-mentioned activities are not well defined, although the role of conserved GDNQ motif of the putative catalytic centre of L protein of few related viruses have been examined. In order to gain insight into the role played by the GDNQ motif of the L protein of Rinderpest virus (RPV), we have examined mutations at each amino acid in this motif of the L protein of Rinderpest virus and tested the biological activity in vivo and in vitro. Site directed mutants were generated and transiently expressed in mammalian cells and were shown to interact with P protein similar to wild type L. The biological activity of mutant L proteins has been tested in an in vitro reconstituted system capable of carrying out cell-free RNA synthesis on synthetic Rinderpest N-RNA template. Further, the role played by individual amino acids has also been defined in vivo using an in vivo minigenome replication/transcription system which indicated the importance of this conserved sequence in viral RNA synthesis.

Preparation and characterization of monoclonal antibodies to nucleocapsid protein N and H glycoprotein of rinderpest virus.

Fifteen stable mouse spleen cell myeloma hybrids (hybridomas) producing monoclonal antibodies to rinderpest virus proteins were produced. The specificity of these monoclonal antibodies was established by radioimmunoprecipitation followed by polyacrylamide gel analysis and immunofluorescence. Nine antibodies were specific for the surface glycoprotein H. All the nine clones showed inhibition of haemagglutination by measles virus. The antibodies from two clones (A7D2 and B2F6) neutralise infectious virus. Six clones produce antibodies reacting with the nucleocapsid protein N. Three antigenic sites designated I-III, with sites I and II partially overlapping, were topographically mapped on the H molecule by competitive binding assay. Similarly, two antigenic sites I and II were delineated on the N protein. The monoclonal antibodies were used to study the antigenic relationships of H and N proteins of rinderpest virus, measles virus and canine distemper virus.