Identification of B- and T-Cell Epitopes of BB, a Carrier Protein Derived from the G Protein of Streptococcus Strain G148

Most conventional vaccines consist of killed organisms or purified antigenic proteins. Such molecules are generally poorly immunogenic and need to be coupled to carrier proteins. We have identified a new carrier molecule, BB, derived from the G protein of Streptococcus strain G148. We show that BB is able to induce strong antibody responses when conjugated to peptides or polysaccharides. In order to localize T and B cell epitopes in BB and match them with the albumin-binding region of the molecule, we immunized mice with BB, performed B and T pepscan analyses, and compared the results with pepscan done with sera and cells from humans. Our results indicate that BB has two distinct T helper epitopes, seven linear B-cell epitopes, and one conformational B-cell epitope in BALB/c mice. Four linear B-cell epitopes were identified from human sera, three of which overlapped mouse B-cell epitopes. Finally, three human T-cell epitopes were detected on the BB protein. One of these T-cell epitopes is common to BALB/c mice and humans and was localized in the region that contains the albumin-binding site. These data are of interest for the optimization of new carrier molecules derived from BB.     

Evaluation of Hemagglutinin Protein-Specific Immunoglobulin M for Diagnosis of Measles by an Enzyme-Linked Immunosorbent Assay Based on Recombinant Protein Produced in a High-Efficiency Mammalian Expression System

Recombinant hemagglutinin (H) of the measles virus (MV) expressed in a mammalian high-expression system based on the Semliki Forest virus replicon was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulin M (IgM) and IgG in patients with acute-phase measles. One hundred twelve serum specimens from 70 patients with measles were analyzed. Case definition was based on a commercial IgM ELISA that utilizes MV-infected cells (MV-ELISA) (Enzygnost; Behring Diagnostics); the clinical criteria of the Centers for Disease Control and Prevention (Atlanta, Ga.); and/or the increase in hemagglutinin test titers, neutralization test titers, and levels of MV-specific IgG whenever paired sera were available. The initial time courses of the IgM signal after the onset of rash are similar in the H- and MV-ELISAs. On days 0 to 19, both ELISAs detected IgM in 67 of 68 (98.5%) sera. Average maximal levels of IgM seem to persist, however, about 10 days longer in the MV-ELISA (up to day 25) than in the H-ELISA (day 15). From days 20 to 29 and 30 to 59, the H-ELISA detected only 64.3 (9 of 14) and 19.2% (5 of 26), respectively, of sera that were IgM positive by MV-ELISA. At least up to day 30, the performance of the H-ELISA seemed to be similar to that reported for commercial ELISAs based on whole MV. Our results demonstrate that MV H-specific IgM can be used to diagnose most measles cases from a single serum specimen collected within 19 days after the onset of rash and that the recombinant protein used in this study is suitable for this purpose.     

Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay

A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5'-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i) mecA gene, conferring methicillin resistance, common to both S. aureus and Staphylococcus epidermidis; (ii) femA gene from S. aureus; and (iii) femA gene from S. epidermidis. This quantitative approach allows discrimination of the origin of the measured mecA signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant S. epidermidis) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions.     

Cloning of the genome of a densovirus and rescue of infectious virions from recombinant plasmid in the insect host Spodoptera littoralis

We have cloned an infectious genome of the Junonia coenia densonucleosis virus (JcDNV) into the bacterial plasmid pBR322. The viral genome could be rescued from the recombinant plasmid pBRJ by transfection of pBRJ DNA to sensitive Spodoptera littoralis larvae. pBRJ DNA produced a typical viral infection and a comparable percentage of larvae became infected following inoculation of equivalent amounts of purified virion DNA or cloned viral DNA. Virions extracted from transfected larvae were indistinguishable from wild-type (wt) virions with regard to their biophysical and biological properties. In particular, rescued virions were as infectious as wt virions and showed identical restriction profiles of their genome. In contrast, subcloning of JcDNV DNA deleted at both extremities of a sequence of ca 250 or ca 100 bp resulted in the inability of the recombinant plasmids to initiate a viral infection. These data suggest that, as for vertebrate parvoviruses, the inverted terminal repeats display essential functions in the rescue process and replicative cycle of densoviruses. This is the first report of the molecular cloning of the infectious genome from an insect parvovirus, and more generally from an invertebrate virus. pBRJ should provide an efficient tool to further define the organization of the JcDNV genome and compare it to other parvoviruses.     

Fate of the type II pneumocyte following tracheal occlusion in utero: a time-course study in fetal sheep

 Tracheal occlusion in utero has been shown to cause accelerated fetal lung growth and is now being considered as a therapeutic modality for pulmonary hypoplasia. We report the effects of tracheal ligation on the surfactant-producing type II pneumocyte population. Three groups of fetal lambs underwent tracheal ligation of 2 weeks’, 4 weeks’ and 6 weeks’ duration, respectively, and all were sacrificed at 136 days’ gestation (9 days pre-term). Nonoperated twins served as controls. The type II pneumocyte population was studied morphometrically using a combination of anti-surfactant protein B immunohistochemistry and computer-assisted stereologic morphometry at light and electron microscopic levels. Single-factor ANOVA was used for statistical analysis. Two weeks of tracheal ligation resulted in doubling of the total lung volume as a result of airspace distension and, to lesser extent, growth of the tissue compartment. With increasing duration of tracheal ligation, there was no additional lung growth. However, more prolonged tracheal occlusion was found to result in significant reduction of the surfactant system, as reflected in the marked decrease of total pneumocyte type II volume (3.14 cm³, 0.95 cm³, and 0.46 cm³, after 2, 4, and 6 weeks of ligation, compared with 5.96 cm³ for controls) and total pneumocyte type II number (13.9 × 10⁹, 3.8 × 10⁹, and 2.4 × 10⁹, compared with 53.2 × 10⁹ for controls). Ultrastructural analysis of the type II cells in obstructed lungs showed vacuolar degenerative changes that, after 6 weeks of ligation, were apparently irreversible. In utero tracheal ligation causes fetal lung hyperplasia, but results in reduction of and injury to the surfactant-producing cell population. Before tracheal occlusion can find widespread clinical application, its pathophysiology needs to be further elucidated. Received: 30 April 1997 / Accepted: 10 July 1997     

Autosomal genetics and Y-chromosome haplogroup L1b-M317 reveal Mount Lebanon Maronites as a persistently non-emigrating population

Currently, there are 18 different religious communities living in Lebanon. While evolving primarily within Lebanon, these communities show a level of local isolation as demonstrated previously from their Y-haplogroup distributions. In order to trace the origins and migratory patterns that may have led to the genetic isolation and autosomal clustering in some of these communities we analyzed Y-chromosome STR and SNP sample data from 6327 individuals, in addition to whole genome autosomal sample data from 609 individuals, from Mount Lebanon and other surrounding communities. We observed Y chromosome L1b Levantine STR branching that occurred around 5000 years ago. Autosomal DNA analyses suggest that the North Lebanese Mountain Maronite community possesses an ancestral Fertile Crescent genetic component distinct from other populations in the region. We suggest that the Levantine L1b group split from the Caucasus ancestral group around 7300 years ago and migrated to the Levant. This event was distinct from the earlier expansions from the Caucasus region that contributed to the wider Levantine populations. Differential cultural adaption by populations from the North Lebanese Mountains are clearly aligned with the L1b haplotype STR haplogroup clusters, indicating pre-existing and persistent cultural barriers marked by the transmission of L1b lineages. Our findings highlight the value of uniparental haplogroups and STR haplotype data for elucidating biosocial events among these populations.Subject terms: Population genetics, Computational biology and bioinformatics     

Studies using a viral challenge and CD8 T cell depletions on the roles of cellular and humoral immunity in the control of an SHIV-89.6P challenge in DNA/MVA-vaccinated macaques

Here, we study immune responses in four DNA/MVA-vaccinated macaques following an SHIV-89.6P challenge and a subsequent CD8 cell depletion. Both post-challenge and post-depletion peaks of viremia contracted with the expansion, or re-emergence, of CD8 T cells. Post-depletion, CD8 cells expanded in the presence of higher levels of neutralizing Ab and CD4 help than post-challenge and had superior maturational characteristics as measured by expression of the anti-apoptotic protein Bcl-2, the IL-7 receptor CD127 and co-production of IFN-gamma and IL-2. Pre-challenge and pre-depletion titers of neutralizing Ab correlated inversely with peaks of viremia and directly with peaks for anti-viral CD4 cells. Thus, our results reveal CD8 cells playing a central role, and neutralizing Ab, a supporting role in SHIV-89.6P control. They also suggest a dynamic relationship between neutralizing Ab, antigen load and anti-viral CD4 cells in the maturation of high-quality anti-viral CD8 T cells.     

Deprivation of sensory inputs to the olfactory bulb up-regulates cell death and proliferation in the subventricular zone of adult mice

The main olfactory bulb (MOB) is the first relay on the olfactory sensory pathway and the target of the neural progenitor cells generated in the subventricular zone (SVZ) lining the lateral ventricles and which migrate along the rostral extension of the SVZ, also called the rostral migratory stream (RMS). Within the MOB, the neuroblasts differentiate into granular and periglomerular interneurons. A reduction in the number of granule cells during sensory deprivation suggests that neurogenesis may be influenced by afferent activity. Here, we show that unilateral sensory deafferentation of the MOB by axotomy of the olfactory receptor neurons increases apoptotic cell death in the SVZ and along the rostro-caudal extent of the RMS. The vast majority of dying cells in the RMS are migrating neuroblasts as indicated by double Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling/PSA-NCAM labeling. Counting bromodeoxyuridine-labeled cells in animals killed immediately or 4 days after tracer administration showed a bilateral increase in proliferation in the SVZ and RMS which was balanced by cell death on the operated side. These data suggest that olfactory inputs are required for the survival of newborn neural progenitors. The greatest enhancement in proliferation occurred in the extension of the RMS located in the MOB, revealing a population of local precursors mitotically stimulated following axotomy. Together, these findings indicate that olfactory inputs may strongly modulate the balance between neurogenesis and apoptosis in the SVZ and RMS and provide a model for further investigation of the underlying molecular mechanisms of this activity-dependent neuronal plasticity.