Gérard Devauchelle

Synthetic Peptides Derived from the Variable Regions of an Anti-CD4 Monoclonal Antibody Bind to CD4 and Inhibit HIV-1 Promoter Activation in Virus-infected Cells

The monoclonal antibody (mAb) ST40, specific for the immunoglobulin complementarity-determining region (CDR) 3-like loop in domain 1 of the CD4 molecule, inhibits human immunodeficiency virus type 1 (HIV-1) promoter activity and viral transcription in HIV-infected cells. To design synthetic peptides from the ST40 paratope that could mimic these biological properties, a set of 220 overlapping 12-mer peptides frameshifted by one residue, corresponding to the deduced ST40 amino acid sequence, was synthesized by the Spot method and tested for binding to recombinant soluble CD4 antigen. Several peptides that included in their sequences amino acids from the CDRs of the antibody and framework residues flanking the CDRs were found to bind soluble CD4. Eleven paratope-derived peptides (termed CM1–CM11) were synthesized in a cyclic and soluble form. All the synthetic peptides showed CD4 binding capacity with affinities ranging from 1.6 to 86.4 nm. Moreover, peptides CM2, CM6, CM7, CM9, and CM11 were able to bind a cyclic peptide corresponding to the CDR3-like loop in domain 1 of CD4 (amino acids 81–92 of CD4). Peptide CM9 from the light chain variable region of mAb ST40 and, to a lesser extent, peptides CM2 and CM11 were able to inhibit HIV-1 promoter long terminal repeat-driven β-galactosidase gene expression in the HeLa P4 HIV-1 long terminal repeat β-galactosidase indicator cell line infected with HIV-1. The binding of mAb ST40 to CD4 was also efficiently displaced by peptides CM2, CM9, and CM11. Our results indicate that the information gained from a systematic exploration of the antigen binding capacity of synthetic peptides from immunoglobulin variable sequences can lead to the identification of bioactive paratope-derived peptides of potential pharmacological interest.

Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family

We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5′ primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full‐length variable sequences belonging to major (VH1, VH2, VH3, Vκ1 and Vκ21) but also small‐sized (VH9, VH14, Vκ2, Vκ9A/9B, Vκ12/13, Vκ23 and Vκ33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope‐derived peptides.

The replication of Hyposoter didymator polydnavirus: Cytopathology of the calyx cells in the parasitoid

Summary— In the ichneumonid wasp Hyposoter didymator, a polydnavirus was detected in the female reproductive tract. With the aim of studying the regulation of polydnavirus replication, the location and the structure of the virus‐producing cells were determined, and the virus replication process was followed inside the ovaries of young adult females observed in light and electron microscopy. The examination of ovaries of pupa females revealed that virus replication is initiated within the calyx just prior to adult emergence. This study revealed that the calyx appears to be a specialized virogenic tissue which also has, during late pupal life, a secretory function that alters the function of virus production.

The densovirus of Junonia coenia (Jc DNV) as an insect cell expression vector

An infectious genome of the Junonia coenia densovirus (Jc DNV) has been recently cloned and sequenced. We investigated the ability of this cloned genome to be used as expression vector by inserting the lacZ gene of Escherichia coli as fusion gene in the major open reading frame (ORF 1) of the viral sequence. The resulting recombinant plasmid designated pBRJlac Z was transfected into insect SPC-SL 52 cells and the expression of beta-galactosidase (beta-gal) was detected qualitatively or quantitatively by using Xgal or ONPG as chromogenic substrates. Western blot analysis revealed that beta-gal was expressed as chimeric capsid-beta-gal polypeptides. This provided evidence that ORF1 codes for structural polypeptides which share a common C-terminal sequence. Construction of plasmids with alterations or deletions in ORF2, 3 or 4, allowed us to implicate nonstructural (NS) functions in viral DNA replication. Deletions in inverted terminal repeats or in NS functions did not abolish expression of capsid polypeptides but reduced it dramatically. Encapsidation of Jlac Z recombinant genome was achieved by trans-complementation with plasmids bearing intact structural and nonstructural functions. Detection of a beta-gal activity in SPC-SL 52 cells following several subcultures post-transfection suggests that Jlac Z recombinant genome could be maintained in an integrative or episomal state.

Design of cassette baculovirus vectors for the production of therapeutic antibodies in insect cells

BACKGROUND Various systems have been described for the expression of recombinant monoclonal antibodies for therapeutical applications. Insect cells offer great advantages with respect to post-translational modifications, stability, yields and applications. OBJECTIVES To construct plasmid cassette transfer vectors in order to express chimeric, humanized or human antibodies in insect cells using baculovirus expression system. STUDY DESIGN Two transfer vectors, pBHuC kappa and pBHuC gamma 1, were designed. They contain a viral promoter (polyhedrin or p10 promoters, respectively), a signal peptide sequence and a human immunoglobulin light chain C kappa gene or heavy chain C gamma 1 sequence, respectively. Restriction sites have been introduced to allow insertion of rearranged variable genes, after amplification by polymerase chain reaction. RESULTS Recombinant baculoviruses expressing complete immunoglobulins have been generated by a double-recombination event between baculovirus DNA and the loaded cassette transfer vectors. CONCLUSION Our genetic cassette approach makes this system a very flexible and convenient one for the rapid production of therapeutic monoclonal antibodies with heavy and light chains of any human isotype. Specific variable regions selected by the antibody phage display technology can be easily transferred in these vectors to obtain a complete antibody.

Evidence for N-glycosylation and ubiquitination of the prolactin receptor expressed in a baculovirus-insect cell system

The molecular mass of the rabbit prolactin receptor (rbPRLR) deduced from cDNA cloning is 66 kDa. However, the molecular mass of the full‐length receptor expressed in the insect Sf9 cells was found to be 94 kDa. In order to explain this discrepancy, we analyzed the possible post‐translational modifications of the PRLR. Sf9 cells were infected with recombinant baculoviruses in the presence of tunicamycin, an inhibitor of N‐glycosylation. Results showed that an additional ≈ 9 kDa of the extracellular domain could be attributed to the N‐glycosylation and another additional ≈ 20 kDa covalent modification occurred in the cytoplasmic part of the receptor. Western blot analysis, using anti‐ubiquitin antibodies, revealed that the rbPRLR was ubiquitinated in its cytoplasmic domain.

Efficient CD4 binding and immunosuppressive properties of the 13B8.2 monoclonal antibody are displayed by its CDR‐H1‐derived peptide CB11

A systematic exploration of the VH2/Vκ12–13 variable domains of the anti‐CD4 monoclonal antibody (mAb) 13B8.2 was performed by the Spot method to screen for paratope‐derived peptides (PDPs) demonstrating CD4 binding ability. Nine peptides, named CB1 to CB9, were identified, synthesized in a cyclic and soluble form and tested for binding to recombinant soluble CD4. Among them, CB1, CB2 and CB8 showed high anti‐CD4 activity. Competition studies for CD4 binding indicated that PDPs CB1, CB8, and the parental mAb 13B8.2 recognized the same complementarity determining region (CDR)3‐like loop region. PDP CB1 was shown to mimic the biological properties of 13B8.2 mAb in two independent cellular assays, demonstrating inhibitory activities in the micromolar range on antigen presentation and human immunodeficiency virus promoter activation. Our results indicate that the bioactive CDR‐H1 PDP CB1 has retained a significant part of the parental 13B8.2 mAb properties and might be a lead for the design of anti‐CD4 peptidomimetics of clinical interest.

Anti-digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties

A gene encoding a single‐chain antibody fragment directed against digoxin (named 1C10 scFv) was cloned in two expression systems. For this purpose, a new baculovirus transfer cassette fully compatible with the procaryotic pHEN vector was constructed. Baculovirus production led to higher yield than did Escherichia coli expression. The procaryotic fragment showed variations in the fine specificity profile but an affinity constant nearly identical to that of the 1C10 Fab, whereas the eucaryotic scFv fragment had a lower affinity with a specificity profile identical to original mAb. The half‐lives of the digoxin:scFv complexes and the global specificity are compatible with therapeutic use of this antibody fragment.

Electron microscopy study of Melolontha poxvirus: The fine structure of occluded virions

Abstract The fine structure of the occluded virions of Melolontha melolontha poxvirus was studied by electron microscopy of ultrathin sections of spherules and of negatively stained virus suspensions isolated from these inclusions. Virions were oval 4000 × 2500 A. The surface of the virus envelope consisted of spherical units 220 A in diameter arranged without apparent symmetry and giving the virion the aspect of a mulberry, characteristic of poxviruses of the vaccinia subgroup. Each virion possessed an eccentric unilaterally concave core containing a substance of high electron opacity and delimited by a three-layered coat 150 A wide. Inside the core a folded ropelike component, electron lucent and 200 A in diameter, lay parallel to the long axis of the particle. Between the core coat and the virus envelope a substance of intermediate density formed a lateral body. A three-dimensional structure and a diagrammatic representation of a particle was constructed from a study of serial sections of virions. Specific staining of DNA followed by enzyme cytochemistry revealed that the viral genome was localized inside the core. Similarly the proteinaceous nature of the substance surrounding the core was demonstrated. The similarities of structure between this virus, other poxviruses of insects and the poxviruses of vertebrates are discussed.

ADAPTATION OF AN INSECT CELL LINE OF SPODOPTERA FRUGIPERDA TO GROW AT 37° C: CHARACTERIZATION OF AN ENDODIPLOID CLONE

SummarySf21 and Sf9 cell lines established from the lepidoptera Spodoptera frugiperda do not display major induction of heat shock proteins when exposed to a temperature of 37°C. After some months of adaptation at 37°C we obtained two new cell lines, Sf21-HT and SF9-HT, which have now been established for several years in our laboratory. The Sf9-HT line displays a slightly shorter doubling time at 37°C than the wild type at 28°C, but cell lethality gives rise to an earlier growth arrest. The composition of total lipid extract from heat-adapted cells reveals a higher sphingomyelin to phosphatidylcholine ratio and a higher percentage of saturated fatty acids, which are expected for the lower membrane fluidity, required for thermotolerance. The cell volume of Sf9-HT is doubled, and by flow cytometry we showed that the DNA content is twice that in the parental cell line. Karyotypic examination of metaphasic cells achieved under epifluorescence microscopy revealed a doubled chromosome number in Sf9-HT.