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.

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.

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.

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 x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.

VIDAS D-dimer: fast quantitative ELISA for measuring D-dimer in plasma.

VIDAS D-dimer (bioMérieux) is a new quantitative ELISA for D-dimer determination designed for the VIDAS automated system. The test contains single-dose, ready-to-use reagents and is completed within 35 min. Quantitative results are obtained from a calibration curve stored in the software of the system and expressed as fibrinogen equivalent units. The two-step capture/tag test relies on two complementary monoclonal anti-D-dimer antibodies, the second one being labeled with alkaline phosphatase. The upper limit of the measuring range is 1000 micrograms/L and the lower detection limit is <50 micrograms/L, which is below the lower limit of the reference interval (68-494 micrograms/L). Reproducibility (CV) within and between runs ranges from 5% to 7%. There is no interference from heparin, bilirubin, hemoglobin, fibrinogen degradation products, or plasma turbidity. Comparison with a conventional ELISA (y) gave good correlation (r= 0.91, n= 579) and comparable results (y= 1.35x - 148, S(y/x)= 750), especially for D-dimer concentrations ranging from 0 to 1000 micrograms/L (y= 1.09x - 10.6, r= 0.88, S(y/x)= 170).

Monoclonal antibodies targeted to alpha-oligonucleotides. Characterisation and application in nucleic acid detection.

The aim of the present study was to test the antigenicity of alpha-deoxyribonucleotides in order to develop a new tool for the detection of nucleic acid sequences for use in diagnostic applications. We describe four monoclonal antibodies (Mabs) which recognize alpha-deoxyribonucleotides. Two were raised against a poly(alpha-dT) sequence and specifically recognized the alpha-dT nucleotide. Two were raised against a sequence containing all four common nucleotides as alpha-nucleotides and, surprisingly, only recognized the alpha-dG nucleotide. For all four Mabs, no cross reactivity was observed with beta-oligonucleotides. These Mabs were reactive with alpha-oligonucleotide sequences whether these sequences were single-stranded or hybridized to DNA or RNA. The four Mabs were tested in a sandwich hybridization assay that consisted of an alpha-oligonucleotide (for target sequence recognition), one of the four Mabs (for recognition of the hybridized alpha-oligonucleotide), and goat anti-mouse antibody conjugated to horse radish peroxidase (HRP) (for detection). One of the monoclonal antibodies, Mab 2E11D7, was directly conjugated to HRP and used in sandwich hybridization to detect PCR fragments of HPV 18 DNA. The sensitivity of this reaction was 1 pg of plasmid DNA containing the HPV 18 fragment. The specificity of the detection was demonstrated using HPV 6/11 and 16 DNA sequences.

Correlation between the reactivity patterns of monoclonal antibodies to distinct antigenic sites on HN glycoprotein and their protective abilities in Sendai (6/94) virus infection.

The relative importance of the host immune response to various antigenic and functional sites on the HN glycoprotein of Sendai (6/94) virus for protection in vivo, was evaluated in mice passively immunized with monoclonal antibodies to HN and then intranasally challenged with infectious virus. Five neutralizing monoclonal antibodies reacting with distinct antigenic sites and exhibiting different reactivity patterns were selected. All of them were able to prevent entirely the growth of virus in the lungs of experimental animals injected with appropriate dilutions of monoclonal antibody. The calculation of correlation coefficients between the reduction of virus in the lungs of immunized mice and the amount of antibody, expressed in terms of hemagglutination inhibition, hemolysis inhibition or neutralizing units, showed a high degree of correlation (r = 0.89) with neutralization and a lack of correlation (r = 0.44) with hemagglutination inhibition. In parallel a minimum threshold value for protection equivalent to 2 x 10(3) neutralizing units per mouse was determined independently of the mechanism(s) by which monoclonal antibodies mediated the neutralization of the infectivity. On the HN glycoprotein of Sendai (6/94) virus we could not individualize a critical site for successful immune recognition by antibodies although the characteristics of an "ideal protective monoclonal antibody" have also been defined.