Emmanuel Drouet

Selection of a Macaque Fab with Framework Regions Like Those in Humans, High Affinity, and Ability To Neutralize the Protective Antigen (PA) of Bacillus anthracis by Binding to the Segment of PA between Residues 686 and 694

ABSTRACT Human anthrax infection cannot always be treated successfully by antibiotics, as highlighted by recent bioterrorist attacks; thus, adjunct therapies are clearly needed for the future. There is a particular need to further develop adjunct therapies that can neutralize secreted toxins, such as antibodies directed towards the 83-kDa protective antigen (PA83). In the absence of human donors, we immunized a macaque (Macaca fascicularis) with PA83 to obtain such antibodies suitable as an adjunct therapy for human anthrax infection. By using bone marrow as a template, we PCR amplified specific Fab-encoding genes and cloned them as an immune library (107 clones). We isolated a high-affinity (equilibrium dissociation constant [KD], 3.4 nM), highly neutralizing (50% inhibitory concentration, 5.6 ± 0.13 nM) Fab (designated 35PA83) from this library by panning. Its epitope was localized by Pepscan analysis between residues 686 and 694 of PA83 and is part of the region which directly interacts with the cell receptor. 35PA83 may thus neutralize the anthrax toxin by competing directly for its receptor. The genes encoding 35PA83 were similar to those of a human immunoglobulin germ line and were assigned to subgroups of human V, (D), or J genes by IMGT/V-QUEST analysis. The 35PA83 framework regions were 92% identical to a representative allele of each subgroup. When compared to framework regions coded by related human germ line genes, only 2 of 74 (VH) or 75 (VK) analyzed amino acids of 35PA83 have different chemical characteristics. A very high degree of identity with human framework regions makes 35PA83 well suited for expression as a whole primatized immunoglobulin G and demonstrates the practicality of using macaque Fabs when immunized human plasma cell donors are not available.

Fever-like thermal conditions regulate the activation of maturing dendritic cells

Fever is one of the most frequent clinical signs encountered in pathology, especially with respect to infectious diseases. It is currently thought that the role of fever on immunity is limited to activation of innate immunity; however, its relevance to activation of adaptive immunity remains unclear. Dendritic cells (DCs) that behave as sentinels of the immune system provide an important bridge between innate and adaptive immunity. To highlight the role of fever on adaptive immunity, we exposed murine bone marrow‐derived lipopolysaccharide (LPS)‐ or live bacteria‐maturing DCs over a 3‐h period to 37°C or to fever‐like thermal conditions (39°C or 40°C). At these three temperatures, we measured the kinetics of cytokine production and the ability of DCs to induce an allogeneic mixed lymphocyte reaction. Our results show that short exposure of DCs to temperatures of 39°C or 40°C differentially increased the secretion of interleukin (IL)‐12p70 and decreased the secretion of IL‐10 and tumor necrosis factor α by maturing DCs. These fever‐like conditions induced a regulation of cytokine production at the single‐cell level. In addition, short‐term exposed LPS‐maturing DCs to 39°C induced a stronger reaction with allogeneic CD4+ T cells than maturing DCs incubated at 37°C. These results provide evidence that temperature regulates cytokine secretion and DC functions, both of which are of particular importance in bacterial diseases.

Micro-RNAs: viral genome and robustness of gene expression in the host

For comparing RNA rings or hairpins with reference or random ring sequences, circular versions of distances and distributions like those of Hamming and Gumbel are needed. We define these circular versions and we apply these new tools to the comparison of RNA relics (such as micro-RNAs and tRNAs) with viral genomes that have coevolved with them. Then we show how robust are the regulation networks incorporating in their boundary micro-RNAs as sources or new feedback loops involving ubiquitous proteins like p53 (which is a micro-RNA transcription factor) or oligopeptides regulating protein translation. Eventually, we propose a new coevolution game between viral and host genomes.

Sequential Use of Paraformaldehyde and Methanol as Optimal Conditions for the Direct Quantification of ZEBRA and Rta Antigens by Flow Cytometry

ABSTRACT A technique was developed with flow cytometry to quantify the two immediate-early proteins ZEBRA and Rta, which are involved in the activation of Epstein-Barr virus replication. We evaluated four monoclonal antibodies on four cell lines (B95-8, RAJI, Namalwa, and P3HR1) with varying levels of expression of these replication-phase antigens. The Namalwa lymphoma cell line was used as a negative control. Four fixation-permeabilization procedures were compared. The preparation of cells with paraformaldehyde and methanol in sequence, and antigen detection with AZ125 and AR 5A9 monoclonal antibodies, were found to be the optimal conditions in these cell lines. Our procedure allowed ZEBRA antigen to be detected in 4.85% of peripheral blood mononuclear cells from a transplant recipient with a lymphoproliferative disease.

Decrease of blood anti-α1,3 Galactose Abs levels in multiple sclerosis (MS) and clinically isolated syndrome (CIS) patients.

The etiology of multiple sclerosis (MS) remains elusive. Among the possible causes, the increase of anti-Neu5Gc antibodies during EBV primo-infection of Infectious mononucleosis (IMN) may damage the integrity of the blood-brain barrier facilitating the transfer of EBV-infected B cells and anti-EBV T cell clones in the brain. We investigated the change in titers of anti-Neu5Gc and anti-α1,3 Galactose antibodies in 49 IMN, in 76 MS, and 73 clinically isolated syndrome (CIS) patients, as well as age/gender-matched healthy individuals. Anti-Gal and anti-Neu5Gc are significantly increased during IMN (p=0.02 and p<1.10-4 respectively), but not in acute CMV primo-infection. We show that, whereas there was no change in anti-Neu5Gc in MS/CIS, the two populations exhibit a significant decrease in anti-Gal (combined p=2.7.10-3), in contrast with patients with non-MS/CIS central nervous system pathologies. Since anti-Gal result from an immunization against α1,3 Gal, lacking in humans but produced in the gut, our data suggest that CIS and MS patients have an altered microbiota or an altered response to this microbiotic epitope.

New insights into HCV replication in original cells from Aedes mosquitoes

BackgroundThe existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines.MethodsFirst, we used purified E1E2 expressing baculovirus-derived HCV pseudo particles (bacHCVpp) so we could investigate their association with mosquito cell lines from Aedes aegypti (Aag-2) and Aedes albopictus (C6/36). We initiated a series of infections of both mosquito cells (Ae aegypti and Ae albopictus) with the HCVsp (Lat strain - genotype 3) and we observed the evolution dynamics of viral populations within cells over the course of infection via next-generation sequencing (NGS) experiments.ResultsOur binding assays revealed bacHCVpp an association with the mosquito cells, at comparable levels obtained with human hepatocytes (HepaRG cells) used as a control. In our infection experiments, the HCV RNA (+) were detectable by RT-PCR in the cells between 21 and 28 days post-infection (p.i.). In human hepatocytes HepaRG and Ae aegypti insect cells, NGS experiments revealed an increase of global viral diversity with a selection for a quasi-species, suggesting a structuration of the population with elimination of deleterious mutations. The evolutionary pattern in Ae albopictus insect cells is different (stability of viral diversity and polymorphism).ConclusionsThese results demonstrate for the first time that natural HCV could really replicate within Aedes mosquitoes, a discovery which may have major consequences for public health as well as in vaccine development.

Structural and functional characterization of the single-chain Fv fragment from a unique HCV E1E2-specific monoclonal antibody

The nucleotide sequence of the unique neutralizing monoclonal antibody D32.10 raised against a conserved conformational epitope shared between E1 and E2 on the serum‐derived hepatitis C virus (HCV) envelope was determined. Subsequently, the recombinant single‐chain Fv fragment (scFv) was cloned and expressed in Escherichia coli, and its molecular characterization was assessed using multi‐angle laser light scattering. The scFv mimicked the antibody in binding to the native serum‐derived HCV particles from patients, as well as to envelope E1E2 complexes and E1, E2 glycoproteins carrying the viral epitope. The scFv D32.10 competed with the parental IgG for binding to antigen, and therefore could be a promising candidate for therapeutics and diagnostics.

Lytic EBV infection investigated by detection of Soluble Epstein-Barr virus ZEBRA in the serum of patients with PTLD

The ZEBRA protein (encoded by the BZLF1 gene), is the major transcription factor of EBV, expressed upon EBV lytic cycle activation. Several studies highlighted the critical role of EBV lytic infection as a risk factor for lymphoproliferative disorders like post-transplant lymphoproliferative disease (PTLD). Here, we use an antigen-capture ELISA assay specifically designed to detecting the circulating soluble ZEBRA (sZEBRA) in serum samples (threshold value determined at 40ng/mL). We retrospectively investigated a population of 66 transplanted patients comprising 35 PTLD. All the samples from a control population (30 EBV-seronegative subjects and 25 immunocompetent individuals with EBV serological reactivation), classified as sZEBRA < 40ng/mL were assigned as negative. At PTLD diagnosis, EBV genome (quantified by qPCR with EBV DNA>200 copies/mL) and sZEBRA were detectable in 51% and 60% of cases, respectively. In the patients who developed a pathologically-confirmed PTLD, the mean sZEBRA value in cases, was 399 ng/mL +/− 141 versus 53ng/mL +/− 7 in patients who did not (p  < 0,001). This is the first report relating to the detection of the circulating ZEBRA in serum specimens, as well as the first analysis dealing with the lytic cycle of EBV in PTLD patients with this new biomarker.