Karim Dorgham

Ismaïl Dogan

Natacha Bitton

Christophe Parizot

Valerie Cardona

Patrice Debré

Oliver Hartley

Dr Guy Gorochov
email: guy.gorochov@psl.ap-hop-paris.fr.

Immunogenicity of HIV Type 1 gp120 CD4 Binding Site Phage Mimotopes

Keywords: CD4bs, CD4 binding site, ScFv, Single-chain variable fragment, PBSM, Phosphate Buffer Saline, IFA, Incomplete Freud Adjuvant

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INTRODUCTION

The Human Immunodeficiency Virus type 1 (HIV-1) interacts with target cells by virtue of a high-affinity interaction between host cell CD4 and the viral envelope glycoprotein (Env), which is formed by cleavage of the glycoprotein (gp)160 precursor into two non-covalently associated polypeptide chains, gp120 and gp41. [START_REF] Willey | Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160[END_REF] The HIV-l envelope is currently the major focus in the design of HIV-1 humoral vaccines and antigen-based immunotherapeutic strategies.

Two functionally and temporally distinct classes of neutralizing antibodies (Abs) are generated against HIV-l in infected humans. Highly strain-specific antibodies are elicited early after initial infection. Later in the course of infection, antibodies capable of neutralizing a wider range of HIV-1 isolates appear, but cross-neutralizing titers remain typically low. [START_REF] Nara | Neutralization of mV-1: A paradox of humoral proportions[END_REF][START_REF] Haynes | Toward an understanding of the correlates of protective immunity to mv infection[END_REF][START_REF] Moog | Autologous and heterologous neutralizing antibody responses following initial seroconversion in human immunodeficiency virus type 1-infected individuals[END_REF] Attempts to induce a broadly protective antibody response against genetically diverse HIV-l isolates have not been successful. [START_REF] Letvin | Progress in the development of an mV-1 vaccine[END_REF][START_REF] Weiss | Variable and conserved neutralization antigens of hu-man immunodeficiency virus[END_REF][START_REF] Robert-Guroff | Spectrum of mv -1 neutralizing antibodies in a cohort of homosexual men: Results of a 6 year prospective study[END_REF][START_REF] Connor | Immunological and virological analyses of persons infected by human immunodeficiency virus type 1 while participating in trials of recombinant gp120 subunit vaccines[END_REF][START_REF] Souza | Evaluation of monoclonal antibodies to mV-1 envelope by neutralization and binding assays: An international collaboration[END_REF][START_REF] Souza | Neutralization of primary HIV-1 isolates by anti-envelope monoclonal antibodies[END_REF] This can be explained in part by continuous in vivo evolution of the virus (viral escape, interclade recombinations, and emergence of new viral isolates), which leads to a high degree of antigenic polymorphism.

Moreover, potential neutralizing epitopes on gp120 are occluded by genetically variable loops, [START_REF] Valenzuela | Neutralizing antibodies against the V3 loop of human immunodeficiency virus type 1 gp120 block the CD4-dependent and -independent binding of virus to cells[END_REF][START_REF] Schreiber | The V3-directed immune response in natural human immunodeficiency virus type 1 infection is predominantly directed against a variable, discontinuous epitope presented by the gp120 V3 domain[END_REF] extensive glycosylation, [START_REF] Polzer | The N-linked glycan g15 within the V3 loop of the HIV-1 external glycoprotein gp120 affects coreceptor usage, cellular tropism, and neutralization[END_REF][START_REF] Kwong | HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites[END_REF][START_REF] Wyatt | The antigenic structure of the mv gp120 envelope glycoprotein[END_REF] and subunit interactions within the trimeric structure of the mature HIV -1 envelope. [START_REF] Poignard | Antibody neutralization of HIV-1[END_REF][START_REF] Parren | HIV-1 antibody-debris or virion?[END_REF][START_REF] Burton | A vaccine for mv type 1: The antibody perspective[END_REF] The poor accessibility of broadly neutralizing epitopes probably explains why human monoclonal antibodies (mAbs) that neutralize primary viral isolates in a broad crossclade manner have proven difficult to isolate. [START_REF] Wyatt | The antigenic structure of the mv gp120 envelope glycoprotein[END_REF] Only five such human mAbs have currently been identified. The epitope of one such antibody, IgG1 b12, has previously been studied using different approaches including gp120 mutagenesis, [START_REF] Pantophlet | Fine mapping of the interaction of neutralizing and nonneutralizing monoclonal antibodies with the CD4 binding site of human immunodeficiency virus type 1 gp120[END_REF] crystallography, [START_REF] Saphire | Crystal structure of a neutralizing human IGG against HIV-1: A template for vaccine design[END_REF][START_REF] Zwick | Molecular features of the broadly neutralizing immunoglobulin G1 b12 required for recognition of human immunodeficiency virus type 1 gp120[END_REF] or phage display technology. [START_REF] Boots | Anti-human immunodeficiency virus type 1 human monoclonal antibodies that bind discontinuous epitopes in the viral glycoproteins can identify mimotopes from recombinant phage peptide display libraries[END_REF][START_REF] Zwick | Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12[END_REF] It has been revealed to be a discontinuous epitope that overlaps the CD4 binding site (CD4bs) on gp120. [START_REF] Burton | A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals[END_REF][START_REF] Burton | Efficient neutralization of primary isolates of mv -1 by a recombinant human monoclonal antibody[END_REF] 2G12 mAb recognizes a glycan discontinuous epitope involving the C3-V4 region of gp120. [START_REF] Trkola | Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1[END_REF][START_REF] Sanders | The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120[END_REF][START_REF] Scanian | The broadly neutralizing antihuman immunodeficiency virus type 1 antibody 2G 12 recognizes a cluster of alpha1-2 mannose residues on the outer face of gp120[END_REF] The antibody fragment Fab X5, which binds to a CD4-induced (CD4i) epitope, was obtained, like IgG 1 b12, after selection of a phage display library against a CD4-gp120-CCR5 complex. [START_REF] Moulard | Broadly cross-reactive HIV-1-neutralizing human monoclonal Fab selected for binding to gp120-CD4-CCR5 complexes[END_REF] Finally, 2F5 [START_REF] Stiegler | A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1[END_REF][START_REF] Muster | A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1[END_REF][START_REF] Al | Neutralization of divergent human immunodeficiency virus type 1 variants and primary isolates by IAM-41-2F5, an anti-gp41 human monoclonal antibody[END_REF] and z13/4el0 [START_REF] Stiegler | A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1[END_REF][START_REF] Zwick | Neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies[END_REF] mAbs bind epitopes on the ectodomain of gp41.

Passive immunization studies in hu-PBL-SCID mice [START_REF] Parren | Protection against HIV-1 infection in hu-PBL-SCID mice by passive immunization with a neutralizing human monoclonal antibody against the gp120 CD4binding site[END_REF][START_REF] Gauduin | Passive immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIV-1[END_REF] and nonhuman primate models 36- 39 have shown that high doses of Env-targeted neutralizing antibodies can prevent infection.

However, no immunogen capable of eliciting broadly neutralizing Abs has been engineered so far. Elucidation of neutralizing epitope structures may offer valuable insights into the design of such potentially important compounds.

The goal of this study was to identify immunogenic structures that elicit a broad specificity, neutralizing humoral anti-HIV-1 response by screening a phage-displayed peptide library on the anti-CD4bs mAb, IgGl b12. Our results confirm and extend previously published data defining consensus sequences for the epitope of this antibody. [START_REF] Zwick | Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12[END_REF] Most importantly, we show that immunization of mice with phage displaying IgG1 b12 mimotopes induces gp120-specific antibodies.

MATERIALS AND METHODS

Antibodies and purified proteins

MAbs IgGl b12, Fl05, [START_REF] Posner | An IgG human monoclonal antibody that reacts with HIV-l/GP120, inhibits virus binding to cells, and neutralizes infection[END_REF] and recombinant proteins HIV-1SF2 gp120, HIV-lMN gp120, HIV-lCM gp120, HIV-lIIIB (LAV) gp120, HIV-lMN gp160 MN, and soluble CD4 (sCD4) were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. Control proteins ferritin, KLH, and BSA were obtained from Sigma.

Phage-peptides

A peptide library displayed on minor coat protein (pIII) was kindly provided by Igor Fisch and Greg Winter, MRC, Centre for Protein Engineering, Cambridge, UK. This library consists of two recombined repertoires of random decamers, joined together through a central five-residue spacer (ALLRY) and fused to the pIII coat protein of the bacteriophage. [START_REF] Fisch | A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage[END_REF] A control phage displaying tag HA1.l (YPYDVPDYA)-pIII fusion was constructed by cloning an appropriate DNA insert into the phage vector fdMED1 42 (kindly provided by I. Fisch) between restriction sites SfiI and NotI. FdMEDl is derived from the fdDOG1 vector. [START_REF] Hoogenboom | Multi-subunit proteins on the surface of filamentous phage: Methodologies for displaying antibody (Fab) heavy and light chains[END_REF] For alanine scanning, DNA inserts bearing mutations were introduced into FdMEDl clones. Phage dsDNA vectors were purified with QIAprep Spin Miniprep Kit (Qiagen, Courtaboeuf, France) from overnight bacterial cultures. DNA vectors were electroporated into Escherichia coli XLI blue (Stratagene, La Jolla, CA). DNA sequencing was performed using an ABI Prism 377 automatic sequencer (Perkin Elmer, Norwalk, CT).

Biopanning

For phage selection, 5-ml polystyrene tubes (Becton Dickinson, Lincoln Park, NJ) were coated overnight at 4°C with 50 µg of protein in 2 ml phosphate-buffered saline (PBS).

Tubes were washed twice with PBS and blocked with PBS-3% low-fat milk (PBSM) for 2 hr at room temperature. Input phage, 10 12 colony-forrning units (cfu) that had previously been blocked in 2 ml of PBSM were added to the tubes and incubated for 4 hr at room temperature. Unbound phage were removed by washing 10 times with PBS 0.1 % Tween 20 (PBST) and 10 times with PBS. E. coli TGl suspension at mid-log phase (OD600 = 0.6) was added directly into each tube and incubated for 1 hr at 37°C with vigorous shaking.

The bacteria were then plated onto LB petri dishes containing tetracycline (15 µg/ml) and incubated overnight at 37°C. Infected bacteria were scraped from plates and collected in 50 ml of 2YT broth, incubated for 2 hr at 37°C, and then diluted 1/500 in 2YT broth and cultured overnight. Phage were then purified and titered as previously described. [START_REF] Fisch | A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage[END_REF][START_REF] Mccafferty | Phage antibodies: Filamentous phage displaying antibody variable domains[END_REF] After each round of biopanning, isolated clones were randomly picked from LB agar plates and grown overnight at 30°C in 96-well plates (ATGC Biotechnologie, Noisy le Grand, France) filled with 200 µl of 2YT/tetracycline. Plates were centrifuged (250 X g, 10 min) and the supernatants used directly for phage enzyme-linked immunosorbent assay (ELISA).

Phage ELISA and competition assays

Ninety-six-well Maxisorp immunoplates (NUNC, Roskilde, Denmark) were coated overnight at 4°C with 0.2 µg of mAb in PBS/well. After three washes in PBS, plates were incubated 1 hr at 37°C with PBSM. Dilutions of phage or culture supernatants (100 µl) were blocked with 100 µl of PBSM and incubated in plates for 1 hr at 37°C. For competition assays, CsCl-purified phage were added to each well in a volume of 100 µl of PBSM.

Soluble gp120 was then added in a 50 µl volume of PBSM to reach final concentrations of 1, 5, or 10 µg/ml and plates were also incubated for 1 hr at 37°C. Plates were then washed three times with PBST followed by three more with PBS and wells were incubated for 1 hr at 37°C with 100 µl of HRP-conjugated anti-M13 (Amersham, Saclay, France; 1/5000 in PBSM). Plates were again washed (three times PBST, three times PBS) and developed with 100 µl of ImmunoPure TMB Substrate Kit (Perbio Science, Bezon, France) according to the manufacturer' s instructions.

Free peptide mimotopes

Mimotope peptides, with N-termini blocked by acetylation and C-termini blocked by amidation, were synthesized by Boc-based solid-phase peptide synthesis on methylbenzhydrylamine resin and purified by reverse-phase high-performance liquid chromatography to reach a purity of at least 95%. Peptide identity was confirmed by mass spectrometry (MALDI/TOF).

Immunization of mice

Five-week-old female C57BL/6 (Iffa Credo, Lyon, France) were used in all experiments.

For intragastric administration, eight animals were fed by delivering to the stomach 10 12 cfu of PEG-purified phage using a blunt steel feeding tube. [START_REF] Delmastro | lmmunogenicity of filamentous phage displaying peptide mimotopes after oral administration[END_REF] Mice received two gavages on day 0 and one at days 7, 14, and 21. Blood samples were collected by retroorbital puncture before each immunization. For intraperitoneal administration, 10 11 cfu CsCl-purified [START_REF] Smith | Libraries of peptides and proteins displayed on filamentous phage[END_REF] phage that had been dialyzed against PBS at 4°C were emulsified with complete Freund adjuvant (CFA) for the first immunization (week 0) or with incomplete Freund adjuvant (IFA) (boosting, weeks 3 and 6). For conjugation to keyhole limpet hemocyanin (KLH), an additional cysteine residue was added when necessary to the C-terminal end of the peptide. Peptides were then bound to maleimide-KLH (Maleimide-activated KLH Conjugation Kit, Sigma) and purified using gel filtration. Mice were immunized with 50 µg of protein in either CFA or IFA. Blood samples were collected both before and 10 days after each injection and serum was harvested by centrifugation and stored at -20°C. Sera from immunized and control animals were subjected to ELISA. Serum dilutions in PBSM were incubated 1 hr at 37°C in 96-well plates previously coated with 40 µl/well of a 3 µg/ml solution of target proteins [gp160, gp120, ferritin, KLH, bovine serum albumin (BSA)].

After washing, bound IgG were revealed with 1/5000 horse-radish peroxidase (HRP)conjugated anti-mouse IgG (Sigma).

Antibody library construction

Total RNA was extracted 47 from 10 8 splenocytes obtained from three mice that had been immunized with 3e5. RNA was reverse transcribed using the Stratagene single-strand synthesis kit according to the manufacturer's instructions. Oligonucleotides (purchased from Eurogentec, Ougree, Belgium) used for library construction are listed in Table 1.

Single-chain variable fragments (ScFvs) were constructed from rescued genes, essentially as described previously. [START_REF] Marks | By-passing immunization. Human antibodies from V-gene libraries displayed on phage[END_REF] Gene segments encoding the variable domains of the heavy (VH) and of the light chain (Vk) antibodies were amplified separately (94°C 40 sec, 55°C 50 sec, 72°C 2 min 30 times, then 3 min at 72°C) using first round polymerase chain reaction (PCR) primers, [START_REF] Dattamajumdar | Rapid cloning of any rearranged mouse immunoglobulin variable genes[END_REF][START_REF] Jones | Rapid PCR-cloning of full-length mouse immunoglobulin variable regions[END_REF] and subsequently ligated together by addition of a gene segment encoding a l5mer amino acid linker (G4S)3. To this end, first round PCR products were purified (PCR purification kit, Qiagen) and reamplified to append DNA sequences encoding portions of the linker at the 3' end of the VH and the 5' end of the VL genes using JH-link: and VKlink primers together with previously used VHMLn and C kappa primers. VH and Vk segments were then assembled in an overlap extension reaction (10 cycles at 95°C 2 min, 55°C 1 min, and 72°C 3 min) using third round PCR primers encoding appropriate restriction sites for cloning into pHEN1 [START_REF] Hoogenboom | Multi-subunit proteins on the surface of filamentous phage: Methodologies for displaying antibody (Fab) heavy and light chains[END_REF] (Table 1). ScFv gene repertoires were transfected in E. coli and rescued as phagemid particles as previously described. [START_REF] Marks | By-passing immunization. Human antibodies from V-gene libraries displayed on phage[END_REF] For selection of gp160-specific antibodies, 10 10 cfu of input phage were incubated for 4 hr at room temperature in 5-ml polystyrene tubes (Becton Dickinson) previously coated overnight with a 2 ml solution of 25 µg/ml of HIV-l gp160. Washing steps and rescue of eluted phage were performed as described. [START_REF] Marks | By-passing immunization. Human antibodies from V-gene libraries displayed on phage[END_REF] After three rounds of biopanning, individual clones were tested by phage ELISA (described above).

RESULTS

Selection of IgGI bI2-specific phage mimotopes

After biopanning using IgGl b12, 54 clones from the pIII library were individually amplified and tested by phage ELISA. All of these clones (Table 2) bind IgGl b12, but do not bind irrelevant proteins (BSA, hemocyanin, and ferritin) or control Abs (IgG1k, an isotype-matched human control antibody of unknown specificity, and Fl05, a human antibody specific for a gp120 epitope that overlaps the CD4bs but distinct from the IgGl b12 epitope [START_REF] Posner | An IgG human monoclonal antibody that reacts with HIV-l/GP120, inhibits virus binding to cells, and neutralizes infection[END_REF][START_REF] Posner | Neutralization of HIV-I by F105, a human monoclonal antibody to the CD4 binding site of gp120[END_REF] ). Figure 1 shows binding analysis of two representative phage clones.

Alignment of the sequences of IgGl b12-specific phage clones enabled us to define an initial consensus sequence M/VQSD, where Q represents an aromatic amino acid (Table 2). This motif is also present in a previously reported IgGl b12-specific peptide. [START_REF] Zwick | Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12[END_REF] To refine the consensus sequence for binding to IgGl b12, we chose to conduct further investigations based on the overrepresented clone 3e5 (LGPVWSDLTC, 22 of 54 clones, Table 2).

Identification of a minimal peptide sequence critical for interaction with IgG1 bI2

To evaluate the contribution made by each residue in the 3e5 sequence (Table 2) toward IgGl b12 binding, we constructed a panel of individual phage clones bearing single alanine substitutions at each position in the mimotope sequence. The capacity of these 3e5 mutants to bind to IgGl b12 was compared to that of unmodified 3e5 phage ELISA. While substitution of amino acids in the initial consensus sequence resulted in a dramatie reduction of specific interaction with IgGl b12, substitution of residues outside this region had less impact, particularly at positions N-terminal to the consensus sequence (Fig. 2).

Molecular evolution of IgG1 bI2 mimotopes

In an attempt to select structures with improved IgGl b12 binding capacity, we undertook the construction of a second-generation library (L2), in which the consensus sequence M/VWSD was fixed at positions 4, 5, 6, and 7, and diversity was introduced into the flanking regions (Table 3). Biopanning of this library led to the selection of two dominant sequences, VLMVWSDEKG and CLLVWSDEAL. After biopanning, [START_REF] Al | Neutralization of divergent human immunodeficiency virus type 1 variants and primary isolates by IAM-41-2F5, an anti-gp41 human monoclonal antibody[END_REF] clones were picked at random for sequence analysis (Table 3). Comparison of these sequences suggests that a small or hydrophobic residue (preferably leucine) is best accommodated at position 2, while a valine is preferred at position 4 and a glutamic acid at position 8. Based on this information, a third-generation library (L3: XLXVWXDEXX) was designed. We chose to randomize position 6 in this library to verify the importance of serine in the initial consensus sequence, M/VQSD, since serine was abundant (27 clones out of 54) but not ubiquitous at this position after selection of the initial library. Most clones selected from this third library included glycine or serine at the first position and a hydrophobic residue (usually leucine or methionine) at position 3. Histidine and leucine were similarly abundant (9 of 28 and 10 of 28, respectively) at position 9. The data also confirm that serine is indeed favored at position 6 (Table 3). We therefore identified G/SLL/MVWSDEL/H as a refined consensus sequence for binding to IgGl b12 (Table 3), and we opted to carry out further studies using clones 3e5, 3r9, and 19 as representatives from selection on the starting library, L2 and L3, respectively. We first compared by ELISA the binding of these first-, second-, and thirdgeneration phage mimotopes to IgGl b12 (Fig. 3). It would appear from these results that the 3r9 and 19 phage mimotopes (second and third generation, respectively) have a higher affinity for IgG1 b12 than 3e5 (first generation).

Phage peptides and gp160 compete for binding to IgG1 b12

As expected, increasing concentrations of gp120 (1-10 µg/ml) inhibited the binding of phage-displayed mimotopes to IgGl b12 (Fig. 4) while KLH (used as an irrelevant control) had no effect. These results indicate that the selected phage-displayed peptides bind to IgGl b12, mimicking the structure of the IgGl b12 epitope on gp120. On the other hand, we did not detect specific inhibition of the interaction of IgGl b12 with HIV-l gp160 by the corresponding free peptides, which were also inactive in an envelope-dependent cell fusion assay [START_REF] Dragic | Methods in Molecular Genetics[END_REF] (data not shown).

Selected phage mimotopes are weakly immunogenic

KLH-peptide conjugates elicited a strong anti-KLH response but did not elicit any gp160specific antibodies in vivo (data not shown). We therefore focused on the immunogenic potential of phage mimotopes. Anti-HIV-l gp160 IgG or IgA were not detected in the sera of mice subjected to intragastric administration of phage (not shown). Mice were subjected to three intraperitoneal injections with phage peptides 3e5, 3r9, and 19 at three weekly intervals. Sera were collected every 10 days and tested by ELISA for the presence of antiphage and anti-HIV-l gp160 antibodies. At the end of the immunization schedule, all animals had raised substantial amounts of anti-phage antibodies, with end point titers reaching at least 10 4 (data not shown). Notably, anti-gp160 antibodies were also detected in most vaccinated animals, with ELISA signals at least 4-fold higher on gp160 than on BSA (Figs. 5 and6). Stronger ELISA signals were seen with the second-and thirdgeneration phage peptides, 3r9 and 19, than with 3e5, the phage peptide clone isolated from the initial library (Fig. 6). Typically, anti-gp160 antibodies were detected 11 days after the first injection; serum titers peaked at day 31, declined thereafter, and became undetectable after 60 days of follow-up (Fig. 5A). As expected, immunization with control phage HA 1.1 elicited an anti-phage response, but anti-gp160 antibodies were not elicited (Fig. 6). The anti-gp160 response elicited by the phage peptides appears to be of broad selectivity, since sera of immunized mice were reactive against envelope from at least five different, unrelated strains of HIV-l viruses: HIV-lSF2 gp120, HIV-lMN gp120, HIV-lCM gp120, HIV-lIIIB (LAV) gp120, and HIV-lMN gp160 MN (Fig. 5B). Despite the presence of anti-gp160 antibodies in the immune sera, we were unable to detect neutralizing activity on HIV-l TCLA (data not shown).

IgG1 b12-like antibody recovered from a phage display library

To further characterize the anti-gp160 antibody response elicited by the phage peptides, we set out to rescue the VH and VL genes from three mice immunized with the 3e5 phage and clone them for phage display. The complexity of the resulting ScFv library was estimated to be at least 2 X 10 5 different clones. After three rounds of biopanning, phage clones were amplified from single colonies picked at random. The amplified phage were then tested by phage ELISA. Clones were considered HIV specific when their phage ELISA reactivity on gp160 MN was at least 5-fold higher than the background signal with BSA in three separate experiments. Only one such clone (phage ScFv5) was identified. A titration assay suggests that ScFv5 has a significantly lower affinity than ScFvb12 for gp160 (Fig. 7). Sequence analysis of phage ScFv5 revealed that the VH and VL CDR3 sequences (GSLDY and QQSNEDPYT, respectively) do not bear any clear homology with the corresponding regions of IgGl b12. [START_REF] Barbas Cf 3rd | Molecular profile of an antibody response to HIV-1 as probed by combinatorial libraries[END_REF] Assuming that ScFv5 was elicited in response to immunization with an IgG1 b12 mimotope, we set out to ascertain whether ScFv5 recognizes the same epitope as IgGl b12, by measuring inhibition of binding of the phage ScFv to gp160 by IgGl b12. We found that gp160 binding of phage ScFv5 and phage ScFvb12, a phage clone expressing the VH and VL genes of IgGl b12 in ScFv format, [START_REF] Bitton | Characterization of T cell-expressed chimeric receptors with antibody-type specificity for the CD4 binding site of HIV-1 gp120[END_REF] was inhibited to a similar extent by IgG 1 b12 (Fig. 8). Hence it is likely that ScFv5 recognizes an epitope similar to that of IgGl b12.

DISCUSSION

Here we report the use of a phage display approach to identify mimotopes of the neutralizing anti-gp160 antibody IgG 1 b12, [START_REF] Burton | A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals[END_REF][START_REF] Burton | Efficient neutralization of primary isolates of mv -1 by a recombinant human monoclonal antibody[END_REF] and we explore their potential for use as phage peptide vaccines.

Panning a phage peptide library on immobilized IgGl b12 revealed a strong four-residue consensus motif MNQSD (where Q represents an aromatic residue, preferably W) (Table 2), and the importance of each residue in the motif was confirmed using an alanine-scanning approach (Fig. 2). Interestingly, a previously described peptide that specifically binds IgGl b12, B2.l (HERSYMFSDLENRCI), [START_REF] Zwick | Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12[END_REF] which was selected from a phage peptide library displayed on pVIII, also contains the four-residue motif (bold letters). In this study, the authors emphasized the importance of an unpaired cysteine residue for binding activity, showing that it enabled the formation of IgG1 b12-binding peptide homodimers to be presented on the phage coat protein via a disulfide bond. Some of the IgGl b12-specific mimotopes described in our study (including 3e5) include an unpaired cysteine residue, but since this is not a constant feature of all specific clones, we would suggest that the formation of homodimers is not essential for the binding activity of the phage mimotopes.

To extend and refine the consensus sequence, we constructed second-and third-generation libraries (L2 and L3), in which the amino acids identified in earlier selection experiments were fixed, and diversity was introduced into flanking positions. Analysis of selected sequences from L2 and L3 libraries led us to identify a refined, nine-residue consensus sequence, G/SLL/MVWSDEL/H. Representative clones selected from first-, second-, and third-generation libraries were then compared in terms of immunogenicity. We show in Figure 6 that mice vaccinated with clone 19 (third generation) exhibited on average a stronger gp160-specific antibody response than mice vaccinated with 3e5 (first generation) or 3r9 (second generation). It should be noted, however, that increased immunogenecity is not necessarily a direct reflection of increased binding affinity for IgGl b12. It could equally relate to more efficient expression on the surface of phage, i.e., more copies of pIII-peptide fusions per phage particle.

While we were able to detect competition between phage peptides and soluble gp160 for binding to IgG1 b12, the free, soluble peptide mimotopes were unable to compete efficiently with gp160 or phage-borne peptide for binding to IgGl b12 (data not shown), and they were inactive at blocking envelope-mediated cell fusion when tested up to high micromolar concentrations (data not shown). Together, these results suggest that the environment in which the peptide is presented may have a major impact on its ability to bind IgG1 b12. Binding of unconstrained peptides with their targets invariably leads to entropic loss, which will lower the binding affinity compared to that of the same peptide that is constrained in a binding conformation. [START_REF] O'neil | Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library[END_REF] It is plausible that the peptides were presented in a constrained form, owing to interactions with pIII, and that the phage clones that were selected during biopanning displayed peptides that were constrained into an IgGl b12-binding conformation, thereby enhancing interaction with the antibody. It was shown that a phage-displayed peptide corresponding to the V3 loop of HIV-l binds anti-V3 loop antibodies and adopts a conformation similar to the antibody-bound free peptide. [START_REF] Jelinek | NMR structure of the principal neutralizing determinant of HIV-1 displayed in filamentous bacteriophage coat protein[END_REF] If pIII did play an important role in the presentation of mimotope peptides in a binding conformation, it could be predicted that only pIII-fused mimotopes would efficiently induce cognate antibodies in vivo. This hypothesis is supported by our observation that while immunization with phage peptide mimotopes elicited gp160-specific antibodies, immunization with peptide mimotopes conjugated to KLH did not. Therefore, it is possible that structural constraints imposed by the phage coat play an important role in the immunogenicity of the selected peptide, and that the phage particle, or at least the pIII protein, should be considered an important element in vaccine formulation.

In contrast to mice immunized with the B2.l mimotope peptide, [START_REF] Zwick | Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12[END_REF] mice vaccinated with IgGl b12 phage mimotopes 3e5, 3r9, or 19 clearly mounted an HIV-specific humoral response (Figs. 5 and6). No experimental data or details of the immunization procedure are provided in the article by Zwick et al. [START_REF] Zwick | Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12[END_REF] and it is possible that differences in the immunization protocol used (phage titer, type of adjuvant) could account for the lack of B2.l-induced antibodies cross-reactive to gp120.

In an attempt to characterize the anti-gp160 antibodies in the immune sera from 3e5vaccinated mice, we attempted to rescue anti-gp160 antibodies by phage display. We identified one phage antibody clone (ScFv5) that shares a common epitope with IgG1 b12 on gp120 (Fig. 8). This result provides further confirmation that immunization with phage mimotopes elicits the production of antibodies with IgGl b12-like specificity.

Despite the presence of anti-gp160 immunoreactivity, the immune sera from the immunized mice did not neutralize laboratory-adapted strains of HIV in vitro (data not shown). This could be explained, at least in part, by the relatively low titer of gp160 cross-reactive antibodies in the sera (Figs. 5 and6). Another potential explanation would be that as previously emphasized, [START_REF] Zwick | Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12[END_REF] 18-residue CDRH3 loops, such as the one borne by IgGl b12, are not produced in mice. [START_REF] Mian | Structure, function and prop-erties of antibody binding sites[END_REF] Therefore, it is plausible that only low-affinity antibodies could be raised in mice against that particular epitope. In any case, our results presented in Figure 7 suggest that ScFv5 has a significantly lower affinity than ScFvb12 for gp160.

The problem of low titer could be addressed by vaccine formulation. A notable consequence of the low number of copies (3-5) of pIII per phage particle is that the amount of phagedisplayed peptide that can be injected into an animal is limited, for example, a large dose of phage (10 14 particles) would represent, at best, a few nanomoles of peptide. A potential solution would be to use purified preparations of peptide fused to soluble variants of pIII protein 58 instead of phage. Such an approach would enable the dose of peptide injected to be significantly increased while retaining what we suspect is an essential constrained conformation imposed by the phage protein. The issue of CDRH3 loop length would need to be addressed by vaccination experiments in nonhuman primates, which would be more likely to raise an immune response including antibodies bearing long CDRH3 loops. Of note, protective immune responses have been successfully induced in macaques vaccinated with phage-displayed HIV-l epitopes. [START_REF] Chen | Protection of rhesus macaques against disease progression from pathogenic SHIV-89.6PD by vaccination with phage-displayed HIV-1 epitopes[END_REF] It would be interesting to test mimotopes of a highly neutralizing epitope such as the one recognized by IgGl b12 in such a setting.

FIG. 1 .FIG. 3 .FIG. 5 .

 135 FIG. 1. Analysis of binding of IgGl b12-selected clones by phage ELISA. (A) Binding to indicated (box) plate-adsorbed antibodies. (B) Binding to plate-adsorbed IgGl b12 and indicated irrelevant proteins (box). In each case (A,B) one representative experiment out of five performed is presented.

Table 1 .

 1 TABLES Oligonucleotides used in construction of murine ScFv library

Table 2 .

 2 Phage clones selected by IgG1 b12

Table 3 .

 3 Molecular evolution of IgG1 b12 mimotopesFIGURES

Dorgham et al.Figure 1 Dorgham et al. Figure 2 Dorgham et al. Figure 3 Dorgham et al. Figure 4 Dorgham et al.Figure 5 Dorgham et al. Figure 6 Dorgham et al. Figure 7 Dorgham et al.Figure 8

ACKNOWLEDGMENTS

This study was supported by the Swiss National Science Foundation (Project 3339-62032-00), Agence Nationale de la Recherche contre le SIDA (ANRS, Paris), INSERM, and Assistance Publique-Hopitaux de Paris.


Catherine Vaquero

Wolf H Fridman,'

Janine Moncuit

Marie-Annick Provost,'

Ernesto Falcoff,'

Maria Jose Gelabert,'

Catherine Neauport-Sautes'~

ISOLATION AND PARTIAL CHARACTERIZATION OF MESSENGER RNA, FROM BINDING FACTOR' MURINE T CELL HYBRIDS, CODING FOR SUPPRESSIVE IMMUNOGLOBULIN G

Poly A RNA has been isolated from a murine T cell hybridoma (TzD4) that spontaneously secretes suppressive immunoglobulin G-binding factor (IgGBF). Translation products, obtained from a rabbit reticulocyte lysate translation system and after injection into Xenopus Zaeuis oocytes, contain material with the biologic activity, the affinity, and the m.w. of murine IgGBF; it suppresses secondary in vitro IgG antibody production in a dose-dependent fashion. The suppressive factor binds to IgG but not to IgM immunoadsorbents and, after mild NaDodSO, treatment, dissociates in NaDodSO, polyacrylamide gels into two peaks at 78 and 40 kD. Translation products from two non-IgGBF-secreting cell lines (BW-5147, a T lymphoma line, and Ae, a fibroblast cell line) fail to exert any suppressive activity. On sucrose gradients, the RNA responsible for the biologic activity was found in one major peak located at 11s. IgGBF synthesized in a cellfree translation system by using poly A RNA and sucrose gradient fractions was also characterized by immunoprecipitation with Fc fragments of [35S]methionine-labeled proteins. On NaDodSO, polyacrylamide gels, it migrates in one peak located at 37 kD. We conclude that IgGBF is coded for by 11s poly A RNA and that no post-translational modifications (other than proteolytic cleavage) are necessary to obtain a biologically active factor with Ig-binding properties.

The production of Ig classes and subclasses by B cells is regulated by T cell factors ( l ) , among which immunoglobulin-binding factors (IBF)3 may play a major role (2). Produced by Fc receptor (FcR)-positive T cells, these factors bind specifically to the Fc portion of Ig and modulate the in vitro production of the corresponding Ig isotype.

Three different isotype-specific T cell factors have been

successively described. First, the IgG-binding factor (IgGBF) produced by Fcy receptor-positive T cells (3) binds specifically to the Fc portion of IgG (4) and suppresses the production of IgG antibodies in secondary in vitro antibody responses (5). Second, the IgE-binding factors (IgEBF) produced by Fct receptor-positive T cells bind specifically to IgE and, depending on their glycosylation. enhance (6) or inhibit (7) production of IgE antibodies. More recently, an IgA-binding factor (IgABF) that binds specifically to I g A and suppresses the I g A In vitro antibody response has been described (8). Thus, these factors share common properties: they bind specifically to a given class of antibody and regulate the production of antibodies of the corresponding class.

During these studies, it became apparent that the production of isotype-specific IBF could be specifically induced by the corresponding Ig isotype. For instance, incubation of T cells with IgE leads to the production of IgEBF (9). In an attempt to obtain a homogeneous and unlimited source of IgGBF, T cell hybrids were prepared between alloantigen-activated T cells and BW-5147 T lymphoma cells (10). These hybrids express FcyR and FcaR and produce a factor(s) that, by all criteria tested (biologic activity, affinity, and m.w.1, appear constitutively similar to T cell-secreted IgGBF (2, 10). Recent experiments have shown that incubation of these T cell hybrids (T2D4) in the presence of IgGl , IgG2, or IgA monoclonal proteins induces the production of high amounts of isotype-specific IBF that suppress IgG1, IgG2, or IgA antibody production, respectively (8,11).

In this paper, we present the isolation and characterization of mRNA coding for a suppressor IBF. We show that the poly A RNA extracted from TzD4 T cell hybrids can be specifically translated, in reticulocyte lysates and in oocytes, into polypeptides having a biologic activity, a n affinity, and a m.w. that are similar to cell-secreted IgGBF. After separation on sucrose gradient, IgGBF mRNA was found in one major peak located at 1 1s.

MATERIALS AND METHODS

an IgGBF-producing T hybridoma line T2D4 of H-2' haplotype (10).

Cell lines. Three different cell lines were used to prepare mRNA:

and two nonproducing lines, BW-5147 used as fusion partner for hybridization (10) and Ag. a fibroblast cell line of H-2' (C3H) haplotype (kfndly provided by Dr. R. Cassingena. Vlllejuif]. To prepare T cell hybrids, spleen cells (95% Thy-1.2-positive. 30% FcyR-positive) from BALB/c (H-2d) irradiated mice (850 R) inoculated 5 days earlier with thymocytes from a B10-BR (H-2*) strain were fused, in the presence of polyethylene glycol, with hypoxanthine-aminopterinthymidine (HAT)-sensitive T lymphoma cells, BW-5147 (H-2'. Thyof HAT, FcyR-positive T cell hybrids were grown in RPMI 1640

1.1. FcyR-negative). as described (1 0). After selection in the presence medium (GIBCO. France) containing streptomycin and penicillin (100 U/ml) and supplemented with 10% fetal calf serum (FCS). The T2D4 cell line expresses FcrR and produces suppressive IgGBF (10). TzD4 and BW-5147 cells were grown in ascitic form in pristanetreated Swiss Nu/Nu mice (Biotem. France).

Preparation ofIgG, Fcfragments. a n d immunoadsorbents. Rabbit IgG was isolated from normal serum by precipitation in the presence of 40% (N&)zS04 followed by ion exchange chromatography on DEAE cellulose (DE 52 Whatman) in 0.02 M phosphate buffer, pH 7.2.

Fc and Fab fragments were obtained by digestion of rabbit I g G in the presence of preactivated papain (enzyme to protein ratio of 1 %) for 2 hr at 37°C in 0.1 M phosphate buffer, pH 8. They were separated from undigested IgG by chromatography on Sephadex Gtein A-coupled Sepharose (Pharmacia. France) as described (12). 100 columns and then purified by affinity chromatography on prolmmunoadsorbents of rabbit IgG and Fab fragments were prepared with rabbit IgG. 7 mg/ml. or Fab fragments, 3 mg/ml, as described by coupling cyanogen bromide-activated Sepharose 4B (Pharmacia) Isolation and sucrose gradlent analysls of M A . Total RNA guanidium thiocyanate and centrifugation of the homogenate was isolated from TaD4, BW-5147, and Ag cells by lysis in 4 M through a 5.7 M cesium chloride cushion as described (13). Poly A RNA was purified on a n oligo-dT column (type 7: PL Biochemical) Milwaukee, WI, USA) (14). Poly A RNA, 250 pg, from TzD4 cells was fractionated on a 15 to 30% sucrose gradient following the procedure described (13). Each of the 18 fractions were ethanol-precipitated, washed, dissolved in 20 pl of sterile triple-distilled water, and kept frozen in liquid nitrogen. Ribosomal RNA, tRNA, and globin mRNA were sedimented in parallel tubes and were used as size markers.

Translatlon of W A . Total poly A RNA and poly A RNA sucrosegradient fractions were translated in a micrococcal nuclease-treated reticulocyte lysate (15). Incubation was performed with 0.5 pl (0.25 %) RNA in a final volume of 12.5 pl, in the presence of 2 to 5 pC1 of I3 Sjmethionine (specific activity, 1000 Ci/mM, Amersham, France).

Total incorporation into proteins was determined in 2-pl samples.

Ten microliters of the radioactive peptides were analyzed on a 0.1 % NaDodS04 linear (10 to 20% polyacrylamide gradient) gel under reducing conditions (16). When reticulocyte lysate translation products were prepared for biological tests, incubation was performed with 4 pl(2 pg) of RNA in a final volume of 100 pl in the absence of or after passage on IgG immunoadsorbents.

[35Sjmethionine. and the translation products were assayed directly Total and fractionated poly A RNA were also microinjected into Xenopus laeuis oocytes as described (13). Approximately 50 ng of RNA were microinjected into each of 20 oocytes. Oocytes were incubated for 40 hr at 20°C in Barth's solution (10 plloocyte). The incubation medium was finally withdrawn and stored frozen at -70°C in 1% bovine serum albumin (BSA) until it was assayed for suppressive activity.

Imrnunopreclpltation. Twenty microliters of [35Sjmethionine-labeled reticulocyte translation products were diluted in the same volume of 1 M KCl. 0.1 M Tris. pH 8.8. 2% Triton X-100 with 2 pl of phenylmethylsulfonyl fluoride (4.5 mg/ml in ethanol) and were incubated for 2 hr at 25OC in the presence of 4 pl of Fc fragment (400 d m l ) with occasional agitation. Then 45 a1 of protein A-Sepharose (Pharmacia CL-4B) (160 mg/ml) pre-equilibrated in the same buffer were added and incubated overnight at 4 T . The sediments were washed five times with 0.5 M KCI. 0.01 M Tris, pH 8.8. 0.5% Triton X-100 and twice with 0.01 M Tris, pH 8.8. Occasionally, samples were submitted to a protein A-Sepharose or Fab-Sepharose clearance for 2 hr at 25°C before the addition of Fc fragments (4 pl) and were then processed following the same procedure. Finally, sediments were suspended in 40 pl of electrophoresis sample buffer (1% Na-All supernatants were then applied to NaDodS04 polyacrylamide ( 10 DodS04. 250 mM 8-mercaptoethanol) (p-ME)) and boiled for 5 min. to 20%) slab gels. After electrophoresis, the gels were subjected to fluorography, dried, and autoradiographed.

Isolation o f IgCEF. Isolation of IgGBF was performed by affinity chromatography by using a batch procedure. Translation products. 100 pl, were incubated with 50 pl of Sepharose 4B coupled with rabbit IgG for 16 hr at 4°C. Effluents were recovered, and the immunoadsorbent was then washed five times with 20 vol of sodium phosphate buffer, 0.02 M. pH 7.0. Material bound was eluted by using three successive incubations with 100 pl of 0.2 M glycine-HC1 buffer, pH 2.8, at 4°C. and was then neutralized with 1 M KHZPO4. Effluents and eluates were dialyzed in phosphate-buffered saline (PBS). pH 7.2. The dilution factor due to the elution procedures was taken into account to evaluate the final dilution of products added to In ultro cultures. To prepare cell-secreted IgGBF, T2D4 cells CUItured in uitro were incubated a t 2 x 10' cells/ml at 37OC in balanced salt solution (BSS) for 2 hr.

(4).

Supernatants were collected and passed on IgG immunoadsorbents as described (4). Acid eluates were concentrated 100-fold (as compared with the starting material) on centriflow CF 25 membranes (Amicon, France), dialyzed in PBS. sterilized by passage through 0.22-pm Millipore filters, and used as source of purified IgGBF. inoculated i.p. 10 days before with 2 X 10' sheep red blood cells Secondary In ultro antibody response. Spleen cells from mice.

(SRBC), were cultivated in Falcon plastic tubes at 7 X 10' cells/ml in RPMI 1640 medium (GIBCO. France) containing 100 U/ml penicillin, streptomycin, and 10% fetal calf serum (FCS). Twenty microliters of SRBC. 2 x 10' cells/ml. were added to the 1-ml cultures at day 0 as described (5). After five days of incubation in a 5% COZ. humidified atmosphere, indirect and direct plaque-forming cells (PFC) were measured by using the local hemolysis technique, as described ( 17), that makes use of guinea pig serum as a source of complement. Indirect plaques were revealed by using a rabbit antimouse IgG antiserum. Translation products. effluents, and acid eluates were added to cell cultures at different dilutions a t day 0. Reticulocyte lysates, incubated in the absence of mRNA. and supernatants from oocytes injected with triple distilled water were used as negative controls.

One-dlmenslonal NaDodS04 polyacrylamide gel electrophorethick) in 0.1% NaDodS04 by using the Laemmli buffer system (18).

sls (PAGE). Electrophoresis was performed on slab gels (1.2 mm

One-hundred microliters of TzD4 poly A mRNA translation products (obtained from reticulocyte lysates) and 100-times concentrated, purified IgGBF from TzD4 cells were treated with 0.1 % NaDodS04 for 20 min at 0°C. After addition of glycerol (1/3 final dilution), samples were applied onto 10-cm high 12.5% polyacrylamide gels and were run at 3 mA/cm of gel in parallel with a mixture of '251-labeled S A .

IgG heavy and light chains, and phenol red. After migration. vertical bands corresponding to each sample were cut and then sliced into 0.45-cm slices. Each slice was homogenized and incubated for 16 hr at 0°C with 0.5 ml of RPMI supplemented with 10% FCS. After centrifugation, the incubation medium was recovered and sterilized through 0.22-pm Millipore filters before addition to cell cultures. Bands corresponding to the labeled markers were cut similarly and were directly counted in a gamma counter (LKB, France).

RESULTS

Presence of biologically active IgGBF in translation products

of poly A RNA from T2D4 cells. Poly A RNA was isolated from a n IgGBF-producing T hybridoma line TzD4 and from two nonproducing lines, BW-5147, the T lymphoma line that was used as a fusion partner for hybridoma preparation, and As, a fibroblast cell line. The suppressive activity of the translation products was tested in secondary in vitro anti-SRBC responses. Translation products were added at day 0 of the spleen cell cultures, and the IgG indirect plaque response was measured at day 5.

In the first set of experiments, rabbit reticulocyte lysates were used as an in vitro translation system, and incubation products were tested for suppressive activity in a 1/100 final dilution. As shown in Figure 1 A, proteins encoded by mRNA from T2D4 cells exerted a strong inhibitory effect on the secondary in vitro antibody response (90% inhibition of the IgG indirect plaque response). In contrast, proteins encoded by mRNA from BW-5147 T lymphoma cells and As cells did not significantly modify IgG antibody production. Reticulocyte lysates, when used alone in a 1/100 final dilution, had no effect on the response.

In the second set of experiments, X . laevis oocytes were used as a translation system and were injected with poly A RNA from T2D4 or BW-5147 cells. Supernatants obtained after a 40 hr incubation period were added at a 1/100 final dilution to spleen cell cultures. As shown in Figure lB, supernatants of oocytes injected with BW-5 147 mRNA did not modify the secondary anti-SRBC response, whereas supernatants of oocytes injected with T2D4 mRNA exerted a significant inhibition of the IgG anti-SRBC plaque response (57% inhibition of the indirect PFC). Indeed, supernatants of water-injected oocytes did not exert any inhibitory effect (Fig. 1B).

Therefore these results show that the in vitro and in vivo translation products of mRNA, isolated from an IgGBF-producing T lymphoma line but not those from two nonproducing cell lines, exert suppressive effects on secondary IgG in vitro antibody responses.

To investigate whether IgGBF was, at least in part, responsible for this inhibition, translation products were incubated with immunoadsorbents of Sepharose 4B coupled with rabbit IgG, and the suppressive activities of the acid eluate, effluent, and starting material were compared. As shown in Figure 2, acid eluates, which contain translation products binding to IgG, were found to have enriched suppressive activity when compared with the starting material. At 1/1000 final dilution, the acid eluate and the starting material inhibited 100 and 46% of the response, respectively, whereas the effluent exerted no significant suppressive effect (25% inhibition of the response). These data show that the suppressive activity present in in vitro translation products is dose dependent and can be enriched after passage on IgG immunoadsorbents. When tested at the same dilutions, acid eluates of control lysates (incubated without mRNA) passed on IgG immunoadsorbents did not significantly modify the response (5 to 15% inhibition). In addition, when translation products were passed on Sepharose 4B coupled with IgM, the suppressive activity was found in the effluent (54% inhibition of the response at a 1/500 final dilution) but not in the eluate (no inhibition of the response at the same dilution): these results indicate that, like cell-secreted IgGBF, the translated suppressive factor(s) does not bind to IgM [data not shown).

In conclusion, these results provide evidence that poly A RNA from T2D4 cells specifically encodes for protein[s) having biologic and affinity properties similar to IgGBF.

The m.w. of suppressive IgGBF synthesized in reticulocytes is similar to that of cell-secreted IgGBF. The M, of the suppressive material encoded by T2D4 mRNA was compared with that of IgGBF secreted by T2D4 cells. For this purpose, acid eluates from IgG immunoadsorbents incubated with serum-free supernatants of T2D4 cells were prepared and used as a source of cell-secreted IgGBF. Before electrophoresis, purified IgGBF and in vitro translation products of T2D4 poly A RNA were treated by mild NaDodSO, conditions (0.1 % NaDodSO,) that did not noticeably alter their biologic activity [Fig. 3A andB, right lane).

After gel electrophoresis of translation products, proteins with suppressive activity dissociated into two peaks, corresponding to M, 78 kD and 40 kD (Fig. 3A). Similarly, IgGBF from TzD4 cells gave two peaks of suppressive activity located at 75 kD and 40 kD (Fig. 3B). Indeed, no significant suppressive activity was found at M, between 50 and 35 kD, if translation products of BW-5147 mRNA were run on gels and were tested under the same conditions [ 1 1 % inhibition of the indirect PFC response).

Characterization o f mRNA encoding for IgGBF. TO better characterize the IgGBF mRNA, T2D4 poly A RNA was fractionated by sucrose gradient centrifugation under nondenaturing conditions, and the biologic activities of each fraction were evaluated after translation into oocytes (Fig. 4) and reticulocyte lysates (Fig. 5). inhibition and had no effect at higher dilutions (1/100 and 1/500; data not shown).

To test whether IgGBF was responsible for suppressive activity, translation products obtained from each fraction of mRNA were passed on IgG immunoadsorbents. When acid eluates were tested for biologic activity at a 1/50 final dilution, the two peaks corresponding to fractions 5 to 9 and 16 were recovered. Again, the inhibition was more pronounced in the first peak (80% inhibition with fraction 7) when compared with the second (45% inhibition with fraction 16).

Translation products obtained from reticulocyte lysates, as well as from their corresponding acid eluates from IgG immunoadsorbents, were also tested for suppressive activity, at a 1/250 final dilution, in secondary in vitro anti-SRBC responses. As shown in Figure 5, two peaks of suppressive activity were found, located in fractions 6 to 9 and 14 to 18. When acid eluates were tested at a 1/1000 final dilution, the first peak still inhibited 30 to 50% of the IgG anti-SRBC response, whereas the second peak no longer exerted suppressive activity (data not shown).

When considered together, these results suggested that mRNA coding for biologically active IgG-binding factor is located principally in fractions 5 to 9 that peak at 11s. Some biologic activity that might be due to aggregates of mRNA was also found in a lesser extent in heavier mRNA fractions.

Immunochemical identification of the translation products connected to 1gGBF immunosuppressive activity. Because IgGBF binds to the Fc portion of IgG, attempts were made to identify translation products of total and fractionated T2D4 mRNA by using immunoprecipitation. For this purpose, the radioactive translation products of reticulocyte lysates incubated in the presence of poly A RNA from IgG-producing cells (T2D4) and nonproducing cells (BW-5147 and As) were immunoprecipitated with Fc fragments of IgG followed by protein A-Sepharose. The preparations were then boiled and run on gels. When these methods were used, the products of the three different messengers gave bands located mainly at M, around 45, 37 to 35, and 20 to 16 kD (Fig. 6). The 45-kD band probably corresponded to actin, because this band was also present in immunoprecipitates performed with anti-actin antiserum (data not shown).

The 45-. 35-. 20-. and 16-kD polypeptides were present in immunoprecipitates from T2D4. BW-5147. and As cells as well: their presence probably reflects nonspecific binding to the immunoadsorbent. The 37-kD polypeptide, however, was only found in the products of TZD4 messengers. After clearance with Fab-Sepharose. only two major bands were observed in TZD4 immunoprecipitates. They were located at M, 45 kD and 37 kD and thus presumably correspond to actin and IgGBF. respectively (Fig. 6).

Subsequently, each fraction from the mRNA gradient was translated in reticulocyte lysates, and the translation products were analyzed by using NaDodS04 PAGE before and after immunoprecipitation with Fc fragments.

A s shown in Figure 7A. the m.w. of the synthesized products increases from the top to the bottom through the gradient showing that even large polypeptides could be efficiently encoded by the heavy mRNA fractions. A s illustrated in Figure 7B. after immunoprecipitation with the Fc fragments, various polypeptides were found the M, of which are similar to those observed after Fc binding of the T2D4 poly A RNA products. The 37-kD polypeptide was seen mostly in fractions 7 and 8. faintly in fraction 6, and it was absent in the other fractions. Of note, these fractions corresponded to those encoding for maximal immunosuppressive activity (Fig. 5). Because the 37-kD polypeptide was a) specifically present in immunoprecipitates of TzD4 translation products and b) found only in immunoprecipitates corresponding to the biologically active fractions of the gradient, these data may indicate that it corresponds to In uftro-translated IgGBF.

DISCUSSION

In oocytes and reticulocyte lysates, poly A RNA extracted from the T2D4 T hybridoma line was found to direct the synthesis of proteins that, in terms of biologic activity, affinity, and m.w., are identical to those of IgGBF secretedby the same cells: namely, translation products were found to exert a profound inhibition of the secondary fn uftro IgG antibody response. The inhibitiory activity was bound to IgG immunoadsorbents and was enriched in the corresponding acid eluates. In NaDodS04 PAGE, suppressive proteins synthesized by mRNA appeared to have the same M, as those of cell-secreted IgGBF. Finally, the suppressive activity was specifically translated by mRNA isolated from TzD4 cells producing IgGBF but not from nonproducing cells. Le.. the BW-5147 fusion partner and the Ag fibroblast line. Thus the conclusion determined from these findings is that poly A mRNA from TZD4 cells encodes for IgGBF.

IgGBF has been characterized as a glycoprotein in which the sugar moiety is apparently not involved in the IgG-binding site (1 9). The results, described herein, showing that IgG-binding suppressive molecules were synthesized in rabbit reticulocyte lysates, confirm this observation and suggest that, similar to other lymphokines, such as interferon-/3 (19a) and -y (C. Vaquero, unpublished data) or interleukin 2 [START_REF] Bleackey | Translation of lymphocyte mRNA into biologically active interleukin[END_REF] and antigen-specific suppressor T cell factor [START_REF] Wieder | Cell-free translation of a biologically active antigen specific suppressor T cell factor[END_REF]. no post-translational modifications are necessary to obtain biologically active

IgGBF.

Translation of fractionated poly A RNA resulted in the biosynthesis of immunosuppressive proteins binding to IgG. In both translation systems, i.e., reticulocyte lysates and oocytes, the bulk of suppressive activity was encoded by 9 to 13s mRNA. In the oocyte translation system, a maximal suppressive activity was obtained with 1 1 s mRNA. Some biologic activity was also encountered to a lesser extent around 26s mRNA. It cannot be excluded that this activity might be encoded by a distinct messenger coding for a larger polypeptide having biologic activity similar to IgGBF. More probably, however, it might correspond to aggregates of the smaller messengers, as has already been observed for other lymphokines [START_REF] Derynck | Human interferon y is encoded by a single class of mRNA[END_REF]23).

After mild treatment that does not significantly alter biologic activity, T2D4 mRNA translation products gave, in NaDodSO, PAGE, two peaks of suppressive activity corresponding to 78 and 40 kD. Peaks at identical m.w. were observed after electrophoresis of TzD4-secreted IgCBF treated with the same conditions. In another set of experiments, radioactive translation products of TzD4, BW-5147, and As mRNA were immunoprecipitated with Fc fragments of IgG and were analyzed by NaDodSO, PAGE after denaturing treatment. The translation products of T2D4 messengers with immunosuppressive activity revealed the presence of a 37-kD polypeptide, which was absent in the products of BW-5 147 and As mRNA that were devoid of biologic activity, thus suggesting that this polypeptide may correspond to IgGBF. Other polypeptides were also immunoprecipitated by Fc fragments, but these were also present in the products of As and BW-5 147 messengers and disappeared after Fab-Sepharose clearance indicating that they may nonspecifically bind to immunoadsorbents. The fact that this 37-kD polypeptide is associated with IgGBF was further substantiated by the results from a n analysis of immunoprecipitates obtained with fractionated mRNA. Fractionated in this manner, it was found only in fractions 6 to 8, which exert profound immunosuppressive effects on the in vitro antibody response.

Because, with NaDodSO, PAGE, suppressive activity dissociates at 78 kD and 40 kD under nondenaturing conditions and with Fc fragments, IgGBF immunoprecipitates at 37 kD after denaturing treatment, the present results suggest that the 78 kD may correspond to dimers of 37-kD polypeptides. These results are in accordance with our previous characterization of radio-labeled IgGBF produced by T lymphoma cells (24) and by alloantigenactivated T cells (1 9) indicating that it contained a major chain at 40 kD that associates, in the absence of 0-ME, into 80-kD dimers. In the human, IgGBF was found to be composed of 43 kD and polymers with higher M, of a 23-kD subunit (25).

Other isotype-specific immunoregulatory T cell factors have been described, namely IgEBF that regulate the IgE response [START_REF] Ishizaka | IgE Binding factors from rat T lymphocytes. In Structure and Function of Fc Receptors[END_REF] and the IgABF that suppresses IgA production (8). With respect to IgE regulators produced by T lymphocytes, the IgE-binding activity appears, after Sepharose G-75 filtration, around 40 and 15 kD, whereas the immunoregulatory activity (suppressive or potentiating), is found only at around 15 kD (27,28). Moreover, IgE-specific suppressor substances produced by a T cell hybridoma were described as having M, in the order of 45 kD [START_REF] Watanabe | Regulation of antibody response In different lmmunoglobulln classes. V. Establishment of T hybrid cell line secreting IgE class-speclfic suppressor factor[END_REF].

TzD4 cells secrete IgGBF and express FcyR. Whether IBF and FcR do or do not represent distinct entities is still a matter of debate. The fact, however, that the M, of murine T cell FcyR ranges between 47 and 70 kD [START_REF] Mellman | mouse Fc receptor through the use of a monoclonal antibody[END_REF]. whereas the M, of immunosuppressive IgGBF is around 37 kD, suggests that they might be different molecules or IgCBF represents part of T cell FcyR.

In conclusion, the present experiments provide evidence that mRNA coding for IgGBF has been isolated from murine T cell hybrids and the first basis for the cloning of this factor has been provided. Upon interaction with Ig classes and subclasses, the same hybrids can be induced to produce IgGIBF, IgG2BF, or IgABF (8, 1 1). Thus this model provides a useful tool to use in a n attempt a) to clone the various IBF and b) to study, at the T cell level, the regulation by Ig isotypes of the expression of the genes coding for the different isotype-specific IBF.

  B). Products added to cell cultures at 1/100 final dilution at day 0. Indirect I g G PFC measured at day 5 (direct PFC response: Ftgure 1 . Effect, on the secondary in vitro anti-SRBC response of translation products obtained from reticulocyte lysates (panel A) and In oocytes panel A, 250 & 20 PFC/lOE cells: panel B, 280 & 32 PFC/lOs cells). Control lysate, reticulocyte lysate incubated in the absence of mRNA: control oocyte supernatant, supernatant of water-injected oocytes.

  Final dilutions ent of the suppressive activity of T2D4 poly A RNA translation products Ftgure 2. Dose-response curve and retention, on IgG immunoadsorbsynthesized in reticulocyte lysates and tested on the secondary tn vitro anti-SRBC response. Products added to cell cultures at day 0. Indirect IgG x----x, effluent: 0. control response. (Direct PFC response: 450 2 48 PFC measured at day 5. x-x, starting material: o"--o. acid eluate: PFC/lOE cells).

  r a t i o n d i s t a n c e ( c m ) of the suppressive activity of TzD, poly A RNA translation products FLgure 3. NaDcdSO, PAGE analysis under mild denaturing conditions (obtained in reticulocyte lysates) (Fig. 3A) and of purified IgGBF (prepared from supernatants of T2DI cells] (Fig. 38) on the secondary In uftro anti-SRBC response. Translation products and IgGBF were run on 12.5% acrylamide slab gels. Material eluted from each of the 20 slices was added to cell cultures at 1/20 final dilution at day 0. Results are expressed as percent inhibition of the indirect IgG PFC response measured at day 5. Indirect responses: panel A, 2700 ir 540 PFC/106 cells: panel B, 3220 ir 108 PFC/lOs cells. Direct responses: panel A. 480 ir 60 PFC/106 cells: panel B, 200 .t 2 5 PFC/106 cells. Right lanes, percent inhibition of the indirect IgG PFC response obtained with 0.1 % NaDodS0,-treated starting material (scale shown on the left of the figure). Molecular weight marker proteins are indicated on the top of the figure.

FLgure 4 .FLgure 5 .

 45 uftro anti-SRBC response of oocyte translation products of TzD4 poly A Immunosuppressive activity associated with the secondary RNA sucrose gradient fractions. Supernatants from oocytes microinjected with each sucrose gradient fraction(x-x) or acid eluates from superdilution at day 0 to cell cultures. Results are expressed as percent inhi-natants passed on IgG immunoadsorbents (0--0) were added at 1/50 bition of the indirect IgG PFC response measured at day 5.The translation products secreted in supernatants of injected oocytes were assayed for biologic activity at a 1 / 50 final dilution. As shown in Figure4, two peaks of suppressive activity could be found: a) a major peak at fraction 7 (1 lS), which exerts 100% inhibition of the IgG anti-SRBC secondary response at a 1/50 dilution and 70% inhibition at a 1/500 dilution (data not shown), and b) a minor peak at fraction 16, which exerts only 30%Fraction number Immunosuppressive activity associated with the secondary in uttro anti-SRBC response of reticulocyte translation products of T2D4 poly A RNA sucrose gradient fractions. Reticulocyte products obtained with each sucrose gradient fraction (x-x) and acid eluates (0-0) from reticulocyte products passed on IgG immunoadsorbents were added to cell cultures at 1/250 final dilution at day 0. Results are expressed as percent inhibition of the indirect IgG PFC response measured at day 5.

of

  TlD4 poly A RNA with Fc fragments as analyzed by NaDodS04 PAGE. Ffgure 6. Immunoprecipitation of reticulocytes translation products [35S]methionine-labeled translation products of poly A RNA from As (lane 1 ) . BW-5147 (lane 2). and TID. cells (lanes 3 and 4) were immunoprecipitated with Fc fragments followed by protein A-Sepharose. before (lanes 1-3) or after (lane 4) clearance with Fab-Sepharose and run on 10 to 20% acrylamide slab gels. Molecular weight marker proteins are shown on the right of the figure. Arrow indicates the 37-kD polypeptide.

Ftgure 7 .

 7 NaDodSO. PAGE analysis (10 to 20% acrylamlde) of 1-1 fractions of TlD4 poly A RNA, before (A) or after (B) immunoprecipitation methionine-labeled reticulocyte translation products of sucrose gradient wlth Fc fragments. The first lane on the left (HaO) represents incubation wlthout mRNA. lanes 5 to 18 represent translation products from the corresponding sucrose gradient mRNA fractions (top to bottom). Translation products from sucrose gradient fractions 1 to 4 showing very low template activity are not presented. Molecular weight markers are indicated on the right of the figure. <. indicates the 37-kD polypeptide.

Acknowledgments. We wish to acknowledge Dr. R. Cassingena (1.R.S.C.-Villejuif) for kindly providing As cells and Dr. M. Stanislawski (1.R.S.C.-Villejuif) for his help in preparing the Fc and Fab fragments.

Recherche Midicale (Grant 650-79-65). Centre National de la Recherche ' This work was supported by grants from lnstitut de la Sante et de la Scientifique (Grant 60-82-501) and Ministere de I'Industrie et de la Recherche (Grant 153). 'Ulm, 75005-