Nadia Piga

The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients.

Mutations in Notch3 cause CADASIL (cerebral autosomal dominant adult onset arteriopathy), which leads to stroke and dementia in humans. CADASIL arteriopathy is characterized by major alterations of vascular smooth muscle cells and the presence of specific granular osmiophilic deposits. Patients carry highly stereotyped mutations that lead to an odd number of cysteine residues within EGF-like repeats of the Notch3 receptor extracellular domain. Such mutations may alter the processing or the trafficking of this receptor, or may favor its oligomerization. In this study, we examined the Notch3 expression pattern in normal tissues and investigated the consequences of mutations on Notch3 expression in transfected cells and CADASIL brains. In normal tissues, Notch3 expression is restricted to vascular smooth muscle cells. Notch3 undergoes a proteolytic cleavage leading to a 210-kDa extracellular fragment and a 97-kDa intracellular fragment. In CADASIL brains, we found evidence of a dramatic and selective accumulation of the 210-kDa Notch3 cleavage product. Notch3 accumulates at the cytoplasmic membrane of vascular smooth muscle cells, in close vicinity to but not within the granular osmiophilic material. These results strongly suggest that CADASIL mutations specifically impair the clearance of the Notch3 ectodomain, but not the cytosolic domain, from the cell surface.

HCV core immunodominant region analysis using mouse monoclonal antibodies and human sera: characterization of major epitopes useful for antigen detection.

Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1–120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1–120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1–120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2–45) allowed the detection of an anti‐HCV core response by all anticore‐positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20–24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29–33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58–65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7–17, 34–39, and 73–86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton × 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected. J. Med. Virol. 56:300–309, 1998.

Mutual Conformational Adaptations in Antigen and Antibody upon Complex Formation between an Fab and HIV-1 Capsid Protein p24

BACKGROUND Elucidating the structural basis of antigen-antibody recognition ideally requires a structural comparison of free and complexed components. To this end we have studied a mouse monoclonal antibody, denoted 13B5, raised against p24, the capsid protein of HIV-1. We have previously described the first crystal structure of intact p24 as visualized in the Fab13B5-p24 complex. Here we report the structure of the uncomplexed Fab13B5 at 1.8 A resolution and analyze the Fab-p24 interface and the conformational changes occurring upon complex formation. RESULTS Fab13B5 recognizes a nearly continuous epitope comprising a helix-turn-helix motif in the C-terminal domain of p24. Only 4 complementarity-determining regions (CDRs) are in contact with p24 with most interactions being by the heavy chain. Comparison of the free and complexed Fab reveals that structural changes upon binding are localized to a few side chains of CDR-H1 and -H2 but involve a larger, concerted displacement of CDR-H3. Antigen binding is also associated with an 8 degrees relative rotation of the heavy and light chain variable regions. In p24, small conformational changes localized to the turn between the two helices comprising the epitope result from Fab binding. CONCLUSIONS The relatively small area of contact between Fab13B5 and p24 may be related to the fact that the epitope is a continuous peptide rather than a more complex protein surface and correlates with a relatively low affinity of antigen and antibody. Despite this, a significant quaternary structural change occurs in the Fab upon complex formation, with additional smaller adaptations of both antigen and antibody.

Generation of monoclonal antibodies to native human immunodeficiency virus type 1 envelope glycoprotein by immunization of mice with naked RNA

The Semliki Forest virus (SFV) vector system is a new approach for in vivo expression of heterologous proteins and can also be used to generate specific immune responses in animal models. HIV-1 envelope glycoprotein produced using the SFV expression system is correctly folded, cleaved, transported to the cell surface and exhibits functional activity. We evaluated a recombinant Semliki Forest virus naked RNA-based immunization protocol for generation of monoclonal antibodies against the HIV-1 envelope glycoprotein. In vitro-transcribed RNA encoding for the SFV replicase complex and Env protein of HIV-1 (HXB2 strain) was injected intramuscularly to mice. This approach elicited an Env-specific antibody response in four mice out of five and a monoclonal antibody, 12H2, directed against gp41 was produced. Our results show that recombinant SFV RNA immunization can potentially be used as a quick and direct method to produce monoclonal antibodies, with the particular advantage that vectored RNA, rather than purified antigen, delivers a complex oligomer produced correctly.

Collection of human biological samples for research purpose: Key challenges and patients’ perspectives

The development and the access to collections of human biological samples is one of the major challenges for health research. In recent years, biological resource centres (BRCs) have developed in such a way that they provide all activities relating to the handling of samples. In this context, France is undoubtedly a pioneering country, because most of the biological collections available were created on the basis of themed research projects, which involved a particular donor phenotype. The round table was an opportunity to emphasise the persistence of some pitfalls particularly in relation to ensuring the consistency of different regulatory pathways. It also gave the opportunity to question and make recommendations on aspects of governance of biological collections and the BRCs, to state the challenges linked to scientific and economic valorisation and to consider the place of patients and the general public. The development of specific education in public health and research is essential to underline that these initiatives are necessary for developing new diagnostic and therapeutic procedures.

Collections d’échantillons biologiques humains pour la recherche : principaux enjeux et conséquences pour le patient ?

Resume La constitution et l’acces a des collections d’echantillons biologiques humains representent des enjeux majeurs pour la recherche en sante. Ces dernieres annees, des centres de ressources biologiques (CRB) se sont developpes de maniere a assurer l’ensemble des activites relatives a la gestion de ces echantillons. Dans ce contexte, la France est sans doute un pays precurseur, ce d’autant que la plupart des collections biologiques disponibles ont ete constituees autour de projets de recherche thematises associant un phenotypage precis des donneurs. La table ronde a ete l’occasion de souligner la persistance de quelques ecueils notamment au regard de la mise en coherence des differents parcours reglementaires. Elle constituait aussi l’opportunite de s’interroger et de proposer des recommandations sur les aspects de gouvernance des collections biologiques et des CRB, de preciser les enjeux relatifs a la valorisation scientifique et economique et de reflechir a la place des patients et de la population generale. Le developpement d’une education a la sante publique et a la recherche est imperatif pour rappeler que ces demarches sont necessaires afin de favoriser le developpement de nouvelles approches diagnostiques et therapeutiques.

Evaluation of two VIDAS ®prototypes for detecting anti-HEV IgG

BACKGROUND High performance anti-hepatitis E virus (HEV) IgG assays are crucial for epidemiology. OBJECTIVE To evaluate the performance of 2 prototypes developed for the VIDAS® automatic system for detecting anti-HEV IgG, one based on the ORF2 antigen (ORF2 prototype) and the other on the ORF2 and ORF3 antigens (ORF2/3 prototype), with reference to the Wantai anti-HEV IgG assay. STUDY DESIGN The sensitivity of each assay was determined by testing 113 blood samples, 63 from immunocompetent and 50 from immunocompromised patients, with a proven HEV infection defined by detecting HEV RNA. Their specificity was assessed with 103 blood samples that the Wantai assay indicated was negative for anti-HEV IgM and IgG, and negative for HEV-RNA. Cross reactivity was assessed using samples that were positive for hepatitis A virus IgG (n=16), hepatitis C virus antibodies (n=15), hepatitis B virus antigen and anti-HBc antibodies (n=16), rheumatoid factor (n=14), and negative for anti-HEV IgG with the Wantai assay. RESULTS The sensitivities in immunocompetent patients were: 95.2% (ORF2), 96.8% (ORF2/3), and 93.6% (Wantai); in immunocompromised patients they were: 66% (ORF2), 72% (ORF2/3), and 68% (Wantai). Both VIDAS prototypes detected low concentrations of anti-HEV IgG. The overall specificity was 100% (ORF2 prototype) and 98.1% (ORF2/3 prototype). Both VIDAS prototypes cross-reacted in five samples (9.6%), mainly those containing HCV antibodies or rheumatoid factor. CONCLUSION Both VIDAS® prototypes performed very well and appear to be suitable for routine detection of anti-HEV IgG.