Engineering antibody heavy chain CDR3 to create a phage display Fab library rich in antibodies that bind charged carbohydrates.

peer reviewedA number of small charged carbohydrate moieties have been associated with inflammation and cancer. However, the development of therapeutic Abs targeting these moieties has been hampered by their low immunogenicity and their structural relationship to self-Ag. We report the design of an Ab repertoire enriched in Abs binding to small charged carbohydrates and the construction of a human Fab phagemid library, "FAB-CCHO." This library combines L chain Ig sequences from human donors and H chain synthetic diversity constructed in key Ag contact sites in CDRs 1, 2, and 3 of the human framework V(H)3-23. The H chain CDR3 has been engineered to enrich the library in Abs that bind charged carbohydrates by the introduction of basic residues at specific amino acid locations. These residues were selected on the basis of anti-carbohydrate Ab sequence alignment. The success of this design is demonstrated by the isolation of phage Abs against charged carbohydrate therapeutic target Ags such as sulfated sialyl-Lewis X glycan and heparan sulfate

Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5′ and/or 3′ end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 × 10(10) and by selecting five unique Fabs against GAPDH antigen

Real-time PCR has advantages over culture-based methods in identifying major airway bacterial pathogens in chronic obstructive pulmonary disease: Results from three clinical studies in Europe and North America

IntroductionWe compared the performance of real-time PCR with culture-based methods for identifying bacteria in sputum samples from patients with chronic obstructive pulmonary disease (COPD) in three studies.MethodsThis was an exploratory analysis of sputum samples collected during an observational study of 127 patients (AERIS; NCT01360398), phase 2 study of 145 patients (NTHI-004; NCT02075541), and phase 2b study of 606 patients (NTHI-MCAT-002; NCT03281876). Bacteria were identified by culture-based microbiological methods in local laboratories using fresh samples or by real-time PCR in a central laboratory using frozen samples. Haemophilus influenzae positivity with culture was differentiated from H. haemolyticus positivity by microarray analysis or PCR. The feasibility of bacterial detection by culture-based methods on previously frozen samples was also examined in the NTHI-004 study.ResultsBacterial detection results from both culture-based and PCR assays were available from 2,293 samples from AERIS, 974 from the NTHI-004 study, and 1736 from the NTHI-MCAT-002 study. Quantitative real-time PCR (qPCR) showed higher positivity rates than culture for H. influenzae (percentages for each study: 43.4% versus 26.2%, 47.1% versus 23.6%, 32.7% versus 10.4%) and Moraxella catarrhalis (12.9% versus 6.3%, 19.0% versus 6.0%, 15.5% versus 4.1%). In the NTHI-004 and NTHI-MCAT-002 studies, positivity rates were higher with qPCR for Streptococcus pneumoniae (15.6% versus 6.1%, 15.5% versus 3.8%); in AERIS, a lower rate with qPCR than with culture (11.0% versus 17.4%) was explained by misidentification of S. pseudopneumoniae/mitis isolates via conventional microbiological methods. Concordance analysis showed lowest overall agreement for H. influenzae (82.0%, 75.6%, 77.6%), due mainly to culture-negative/qPCR-positive samples, indicating lower sensitivity of the culture-based methods. The lowest positive agreement (culture-positive/qPCR-positive samples) was observed for S. pneumoniae (35.1%, 71.2%, 71.2%). Bacterial load values for each species showed a proportion of culture-negative samples with a load detected by qPCR; for some samples, the loads were in line with those observed in culture-positive samples. In the NTHI-004 study, of fresh samples that tested culture-positive, less than 50% remained culture-positive when tested from freeze/thawed samples. In the NTHI-004 study, of fresh samples that tested culture-positive, less than 50% remained culture-positive when tested from freeze/thawed samples.DiscussionReal-time PCR on frozen sputum samples has enhanced sensitivity and specificity over culture-based methods, supporting its use for the identification of common respiratory bacterial species in patients with COPD

Varicella-zoster virus induces apoptosis in cell culture

Apoptosis is an active mechanism of cell death which can be initiated in response to various stimuli including virus infections. In this work, we demonstrate that lytic infection by varicella-zoster virus (VZV), a human herpesvirus, is characterized by nuclear fragmentation of DNA into oligonucleosomal fragments and by chromatin condensation. In vitro, VZV-induced cell death is actually mediated by apoptosis. The mechanisms developed by cells to protect themselves against apoptosis could be one of the parameters allowing the establishment of virus latency. In the case of VZV, which can remain latent in sensory ganglia, we have not yet identified a cellular or viral protein which could play this protective role, since the observed apoptosis mechanism seems to be independent from Bcl-2, the most frequently described inhibitor of apoptosis

Etude ultrastructure de l'organisation du nucléoïde du virus de la varicelle et du zona

peer reviewe

Intracellular distribution of the ORF4 gene product of varicella-zoster virus is influenced by the IE62 protein

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for genes of heterologous viruses, The major regulatory immediate-early protein of VZV (IE62) is a transactivator of VZV gene expression, In transfection assays, IE4 has been shown to enhance activation induced by IE62, To investigate the functional interactions underlying this observation, indirect immunofluorescence studies were undertaken to determine whether IE62 could influence IE4 intracellular localization in transfected cells, In single transfections, IE4 was predominantly found in cytoplasm, In cotransfection with IE62, the IE4 localization pattern was altered, with nuclear staining predominating over cytoplasmic staining, This effect was specific to the IE62 protein since the gene products of ORF63 and ORF61, which are also regulatory proteins, did not influence IE4 distribution, The use of IE62 mutants indicated that IE62 influence is independent of its transactivation function and that the integrity of regions 3 and 4 is required, IE62 remained nuclear whether IE4 was present or not, These observations underline differences in the regulation of gene expression between VZV proteins and their herpes simplex virus type 1 homologues, In infected cells, IE4 was only sometimes found to colocalize with IE62 in nuclei, This observation suggests that when all VZV proteins are present, complex interactions probably occur which could diminish the influence of IE62

Lessons to be learned from varicella-zoster virus

Varicella-zoster virus (VZV) is an alphaherpesvirus responsible for two human diseases: chicken pox and shingles. The virus has a respiratory port of entry. After two successive viremias, it reaches the skin where it causes typical lesions. There, it penetrates the peripheral nervous system and it remains latent in dorsal root ganglia. It is still debatable whether VZV persists in neurons or in satellite cells. During latency, VZV expresses a limited set of transcripts of its immediate early (IE) and early (E) genes but no protein has been detected. Mechanisms of reactivation from ganglia have not been identified. However, dysfunction of the cellular immune system appears to be involved in this process. The cell-associated nature of VZV has made it difficult to identify a temporal order of gene expression, but there appears to be a cascade mechanism as for HSV-1. The lack of high titre cell-free virions or recombination mutants has hindered so far the understanding of VZV gene functions. Five genes, ORFs 4, 10, 61, 62, and 63 that encode regulatory proteins could be involved in VZV latency. ORF4p activates gene promoters with basal activities. ORF10p seems to activate the ORF 62 promoter. ORF61p has trans-activating and trans-repressing activities. The major IE protein ORF62p, a virion component, has DNA-binding and regulatory functions, transactivates many VZV promoters and even regulates its own expression. ORF63p is a nuclear IE protein of yet unclear regulatory functions, abundantly expressed very early in infection. We have established an animal model of VZV latency in the rat nervous system, enabling us to study the expression of viral mRNA and protein expression during latency, and yielding results similar to those found in humans. This model is beginning to shed light on the molecular events in VZV persistent infection and on the regulatory mechanisms that maintain the virus in a latent stage in nerve cells