Gangliosides, Ab1 and Ab2 antibodies

Molecular Immunology 44 (2007) 2915–2922

Gangliosides, Ab1 and Ab2 antibodies III. The idiotype of anti-ganglioside mAb P3 is immunogenic in a T cell-dependent manner Alejandro L´opez-Requena a,b , Marco Bestagno b , Cristina Mateo de Acosta a , Michela Cesco-Gaspere b , Ana Mar´ıa V´azquez a , Rolando P´erez a , Oscar R. Burrone b,∗ b

a Department of Antibody Engineering, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba Molecular Immunology Group, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy

Received 14 November 2006; accepted 14 January 2007 Available online 20 February 2007

Abstract P3 mAb is an IgM monoclonal antibody specific for N-glycolyl-containing gangliosides. The immunogenicity of the P3 idiotype has been previously described by immunizing syngeneic BALB/c mice with the purified murine IgM or the mouse-human chimeric IgG antibody. In the present work we study the antibody response against the idiotype of P3 mAb through immunization with DNA. We used small immune proteins (SIP) consisting on the idiotype in the scFv format, covalently linked to ␥1CH3, the self-dimerizing domain of murine IgG1. SIPs were previously shown to be appropriate to induce specific anti-idiotypic responses. By gene gun immunization, a polyspecific response was occasionally generated, particularly with the P3 idiotype. A single shot of DNA was sufficient to induce a strong and long-lasting anti-P3 idiotype response. In addition, by delivery of the same DNA construct with a recombinant adeno-associated virus the unique immunogenicity of the P3 idiotype was demonstrated. The requirement of T cells in the anti-P3 idiotype response was indicated by the lack of P3-specific anti-idiotypic antibodies following immunization of both, allogeneic C57BL/6 and athymic BALB/c mice. © 2007 Elsevier Ltd. All rights reserved. Keywords: Idiotype; SIP; rAAV; Immunogenicity; Anti-idiotypic antibodies

1. Introduction The immunogenicity of autologous immunoglobulins is not a frequently described phenomenon. Both the IgM isotype (Reitan and Hannestad, 1995, 2001, 2002) and somatic hypermutations (Zaghouani et al., 1992; Eyerman and Wysocki, 1994; Cao et al., 1995; Eyerman et al., 1996; Wysocki et al., 1998; Zhang et al., 2001) have been invoked as important factors for this behavior. It was also demonstrated that some peptides derived from the heavy chain variable region (VH) can be immunogenic and even induce an antitumor response (Lou et al., 2004). We previously obtained a monoclonal antibody (mAb), named P3, which is specific for N-glycolyl (NeuGc)-containing gangliosides (V´azquez et al., 1995). The expression in human tumors of this variant of the sialic acid, which is absent from

∗

Corresponding author. Fax: +39 040 226555. E-mail address: [email protected] (O.R. Burrone).

0161-5890/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2007.01.010

normal tissues (Varki, 1992; Watarai et al., 1995), has been extensively described (Marquina et al., 1996; Malykh et al., 2001; Koda et al., 2003). The immunization with anti-idiotypic antibodies (Ab2) as a way of inducing anti-ganglioside antibody responses is a strategy currently used for generating an immune response in cancer patients targeting these antigens (Bhattacharya-Chatterjee et al., 2000; Fern´andez et al., 2003). 1E10 is an anti-idiotypic mAb (Ab2) specific for P3 (V´azquez et al., 1998) that has been used in clinical trials with patients of melanoma (Alfonso et al., 2002) and breast cancer (D´ıaz et al., 2003; Guthmann et al., 2006). P3 is able to induce a very strong anti-idiotypic Ab2 response in syngeneic BALB/c mice, even when administered without carrier proteins or adjuvants (V´azquez et al., 1998). Immunogenetic analysis of P3 and 1E10 mAb variable regions (P´erez et al., 2001) as well as the selection of peptide mimotopes from phage libraries (P´erez et al., 2002; Lopez-Requena et al., 2007a), led to the identification of some key basic residues on P3 H-chain CDRs 1 and 3 that seemed to be complementary with a motif

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of acid residues on different Ab2 specific for P3 mAb (P´erez et al., 2001; Lopez-Requena et al., 2007b). The importance of those residues both for ligand binding and immunogenicity of P3 mAb was demonstrated (Lopez-Requena et al., 2007a). P3 is a germ-line gene-encoded IgM (P´erez et al., 2001). The IgM isotype does not seem to be determinant for its immunogenicity. In fact, other anti-ganglioside IgM tested were unable to induce an Ab2 response in the absence of adjuvants (V´azquez et al., 1998). In the present work, we use DNA immunization and small immune proteins (SIPs) (Li et al., 1997) for assessing the contribution of constant regions for the immunogenicity of the P3 idiotype. SIPs are bivalent proteins based on scFv fragments, where the two variable regions of the H and L chains, VH and VL, respectively, are connected with a tetrapeptide to the murine or human self-dimerizing ␥1CH3 domain or the corresponding domains of other isotypes. SIPs can also be expressed in the membrane-bound format using, for instance, the human ␧CH4 domain (Bestagno et al., 2001). Here, we investigated the immunogenicity of P3 idiotype by DNA immunization either by gene gun or by recombinant adenoassociated virus (rAAV) infection. The requirement of T cell activation for the anti-idiotypic response was demonstrated.

Sweden) and analyzed by SDS-PAGE under reducing conditions. The specificity of the purified antibodies was confirmed by enzyme-linked immunoadsorbent assay (ELISA) and protein concentration estimated by optical density at 280 nm. 2.4. Primers

2. Materials and methods

For the introduction of the appropriate restriction sites for cloning into SIP vectors, the following oligonucleotides were used: for V␬ 5 region (scFv N-terminal): P3 mAb and its mutants: 5 -ACAG GTG CAC TCG GAC ATT GTG ATG ACC3 ; 1E10 mAb: 5 -ACTG GTG CAC TCT GAT ATC CAG ATG ACA-3 . For V␬ 3 : P3 and 1E10 mAbs: 5 -CTACC ACT AGT GCT GCC TTT TAT TTC CAG TTT GG-3 . For VH 5 region: P3 mAb: 5 -CATA C TCG AGC AAA GGA CAG GTG CAG CTG AAG GA-3 ; 1E10 mAb: 5 -ATAC C TCG AGC AAA GGA CAG GTT CAG CTG CAG CA-3 . For VH 3 region (scFv C-terminal): P3 mAb: 5 -AGAT TCC GGA AGA GAC AGT GAC CAG AGT-3 ; 1E10 mAb: 5 -AGT TCC GGA GGA GAC TGT GAG AGT GGT-3 . For the transfer of the whole SIP cassette to the pAAV vector, a BglII site was introduced with a linker at the C-terminal end of the murine ␥1CH3 domain: 5 -CT AGA GCAT AGA TCT TCAG A-3 ; 5 -T CGTA TCT AGA AGTC TGA TC-3 .

2.1. Cells

2.5. Vectors

Sp2/0 and HEK293 cells from American Type Culture Collection (ATCC) (Rockville, MD) were cultured in RPMI 1640 or Dulbecco’s modified Eagles medium (DMEM) (Gibco, Paisley, UK), respectively, supplemented with 10% heat inactivated fetal calf serum (FCS) (Gibco), gentamycin (50 ␮g/mL), and 2 mM l-glutamine. Sp2/0 cells were used for membrane-SIP (memb-SIP) expression and for stable expression of soluble SIPs. HEK293 cells were used for rAAV transient infection and secretion of soluble SIPs. For the selection of stable Sp2/0 SIP-secreting or memb-SIP-displaying transfectomas, RPMI 1640 medium containing 10% FCS and geneticin (G418) at 400 ␮g/mL was used as selective medium.

The pUT-SEC vector for the construction of the scFv fragment has been previously described (Li et al., 1997). The pcDNA3-␥1CH3 (murine) (Li et al., 1997) and pcDNA3-␧CH4sEMPD-TM-C (Bestagno et al., 2001) vectors, both described elsewhere, are for the expression of soluble SIPs or memb-SIPs, respectively. sEMPD stands for short external membrane proximal domain; TM for transmembrane region; C for cytoplasmic tail. The pAAV-MCS vector for the cloning and expression of the secreted SIPs on the rAAV was purchased from Stratagene (La Jolla, CA). pMOS-Blue vector for cloning and sequencing of antibody variable regions was purchased from Amersham Pharmacia (UK).

2.2. Animals

2.6. SIP construction and expression

Female BALB/c, athymic BALB/c (Foxn1nu /Foxn1nu ) and C57BL/6 mice, 6–8 weeks old, were purchased from Harlan (Milan, Italy). Animals were housed and bred in a barriermaintained room according to standard animal care and use guidelines.

PCR amplifications were performed on the variable regions of P3, 1E10 (Lopez-Requena et al., 2003) or P3 H-CDR R → S mutant (Lopez-Requena et al., 2007a) antibodies cloned into pMOS-Blue vector. Amplified antibody variable regions with the suitable restriction sites and cloned into pMOS-Blue vector were sequenced, digested and sequentially cloned into the pUTSEC vector, digested XhoI/BspEI for the VH or ApaLI/SpeI for the V␬. A fragment HindIII/BspEI comprising the scFv fragment with a leader peptide was then transferred into the SIP pcDNA3based vectors equally digested. Sp2/0 cells were transfected by electroporation as previously described (Bestagno et al., 2001). The selection of stable SIP-secreting clones was done by ELISA. Microtiter plates (High binding, Costar, Cambridge, MA) were coated with goat anti-mouse IgG (␥-chain specific) antibody (Sigma, St. Louis, MO) and as second antibody a horse radish

2.3. Ganglioside and chimeric antibodies GM3(NeuGc) ganglioside was kindly provided by Dr. L.E. Fern´andez, Vaccine Department, Center of Molecular Immunology (Havana, Cuba). Chimeric P3, 1E10 (Lopez-Requena et al., 2003) and P3 HCDR mutant (Lopez-Requena et al., 2007a) antibodies (human IgG1, ␬) were purified from transfectoma culture supernatants by Protein-A Affinity Chromatography (Pharmacia, Uppsala,

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peroxidase (HRP)-conjugated goat anti-mouse IgG (␥-chain specific) antibody (Pierce, Rockford, IL) was used. Reaction was developed with the 3,3 ,5,5 -tetramethylbenzidine (TMB) reagent and read at 450 nm. Stable memb-SIP-displaying transfectomas were detected by FACS with a FITC-conjugated goat anti-human IgE (␧-chain specific) antibody (KPL, Kirkegaard and Perry Laboratories, Gaithersburg, MD), and also by incubation with the respective chimeric anti-idiotypic antibody followed by FITC-conjugated goat anti-human IgG (␥-chain specific) antibody (KPL). Flourescence analysis was performed with a FACScalibur (Becton Dickinson, Mountain View, CA) and the CellQuest software. Stable transfectomas expressing the P3- and 1E10-SIPs, as well as transfectomas displaying the respective membrane versions were obtained. 2.7. rAAV preparation A BglII site was introduced with an oligonucleotide linker after the murine ␥1CH3 domain into the XbaI-digested pcDNA3 vector, and the whole SIP fragment was then cloned into the HindIII/BglII-digested pAAV-MCS vector. rAAV particles pseudotyped with the capsid serotype 8 were obtained at the AAV vector unit of the International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy. Virus titration was performed by real-time PCR. Preparations had approximately (2–4) × 1011 units/mL. For checking SIP expression and functionality by ELISA, HEK293 cells were infected with different multiplicities of soluble SIP-coding rAAV and supernatants taken after 72 h.

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in both legs 3 × 1010 viral particles. Serum collection was done 30 days after the infection. 2.9. Antibody binding assays Binding of SIPs to GM3(NeuGc) ganglioside was determined using an ELISA assay as previously described (Alfonso et al., 1995; Lopez-Requena et al., 2007a), with the supernatants from stable transfectomas of from rAAV-infected cells, and a HRP-conjugated goat anti-mouse IgG (␥-chain specific) (Pierce) as second antibody. Ab1–Ab2 interaction was determined by coating the microtiter plates with 10 ␮g/mL of the respective chimeric antibodies, blocking with PBS-Tween 20 0.05%-BSA (bovine seroalbumin) 1% (PBS-T-BSA), and the same secondary antibody. To measure anti-SIP reactivity in sera from immunized mice, the microtiter plates were coated with 10 ␮g/mL of the chimeric antibodies or human IgG purified from normal serum (Sigma). After blocking the plates with PBS-T-BSA, 1:100 diluted serum samples were added and incubated for 2 h at 37 ◦ C, and alkaline phosphatase-conjugated anti-mouse IgG (Pierce) was used as second antibody. Some chosen sera were also tested for reactivity with memb-SIPs, by FACS. Memb-SIP-displaying transfectomas were incubated with 1:100 diluted sera in PBS-BSA 3%. Bound antibodies were detected with a FITC-conjugated goat anti-mouse IgG (␥-chain specific) antibody (KPL). Three samples of each experiment were tested and the coefficient of variation was <10% for all assays. Background values of absorbance were less than 0.1.

2.8. Mice immunization 3. Results The antibody response induced in mice by the SIPs was determined by DNA immunization, either by gene gun or rAAV infection. For gene gun experiments, BALB/c, athymic BALB/c and C57BL/6 mice were immunized intradermally by three shots at 2-week intervals with the SIP constructs. Mice abdominal areas were shaved and 1 ␮m gold particles carrying 1–3 ␮g DNA were injected at 400 psi using the BioRad gene delivery device (Biorad, Hercules, CA). Sera were collected 10 days after the last shot. Additional BALB/c groups received only one shot, and sera were extracted 10, 38 and 101 days after. rAAV infection of BALB/c mice was performed by inoculating intramuscularly

The idiotypes of P3 and P3 mutants assembled as scFvs in the murine SIP format were expressed and tested for their binding activity by ELISA, coating plates with the GM3(NeuGc) ganglioside or the chimeric mouse-human antibodies to ensure only recognition of the idiotypes, thus avoiding interference of constant regions. Only P3-SIP recognized the GM3(NeuGc) ganglioside, while all the P3 H-CDR mutant-SIPs did not (Fig. 1A). The P3- and the P3 mutant-SIPs, with the exception of R31 → S, also recognized the anti-P3 chimeric 1E10 antibody (Fig. 1A). Conversely, 1E10-SIP recognized the chimeric

Fig. 1. Reactivity of small immune proteins (SIPs). ELISA plates were coated (A) with purified GM3(NeuGc) ganglioside or chimeric 1E10 antibody or (B) with chimeric P3 antibody. Reactivity was determined by adding culture supernatants from SIP-producing transfectomas and using an HRP-conjugated goat anti-mouse IgG (␥-chain specific) antibody.

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P3 antibody (Fig. 1B). Transfectomas expressing the membrane bound forms of SIPs with the idiotypes of P3 or 1E10 were also recognized by cytofluorimetry by antibodies 1E10 or P3, respectively (not shown). These results show that the SIP versions of P3, 1E10 and P3 mutant antibodies retain their original binding properties. In order to test the ability of the different SIP constructs to induce specific anti-idiotypic antibodies in vivo, animals were immunized by gene gun with plasmid DNA encoding the different constructs. Sera were tested by ELISA against the different chimeric antibodies and human IgG as control. Surprisingly, one out of three animals immunized with P3-SIP produced antibodies that were not specific, but showed reactivity against all antibodies, including polyclonal human IgG (Fig. 2A). In contrast, animals immunized with the 1E10-SIP, induced a highly specific anti-idiotypic response (Fig. 2B). However, when tested by FACS on cells expressing the P3-memb-SIP or the 1E10memb-SIP (both of human ␧-isotype), the anti-P3 as well as the anti-1E10 sera behaved quite specifically, reacting only with the transfectomas expressing the P3 or the 1E10 idiotypes, respectively (Fig. 3). This result suggested that P3, but not 1E10, can induce in addition to the anti-idiotypic one, a response that also recognizes the human ␥ heavy constant regions, as confirmed by the reactivity against polyclonal human IgG in the ELISA. However, this kind of polyspecific response was occasionally detected (with low titers) also for animals immunized with the 1E10-SIP or other SIPs (data not shown) DNA, although it was clearly more frequent with P3-SIP. When animals were immunized with the SIP construct DNA corresponding to the P3 H-CDR mutants all of them developed anti-idiotypic antibodies against the respective immunizing idiotype, that also recognized the wild type chimeric P3 antibody, but rarely reacted with the human IgG (Fig. 4). There were not significant differences in antibody titers between sera corresponding to the P3-SIP and those from the four mutants (data not shown). In contrast to what has been previously observed with

Fig. 2. IgG response in BALB/c mice immunized by gene gun with P3-SIP (A) or 1E10-SIP (B) DNA. Sera were taken 10 days after the third shot and assayed by ELISA at 1:100 dilution against the chimeric P3 and 1E10 antibodies and human IgG purified from normal serum. Reactivity was determined using an HRP-conjugated goat anti-mouse IgG (␥-chain specific) antibody.

animals immunized with the purified protein (Lopez-Requena et al., 2007a), sera from animals immunized with the SIP construct DNA of the R31 → S mutant produced antibodies recognizing the P3 idiotype. We confirmed these results with sera from DNA or protein-immunized animals by cytofluorymetry on cells displaying the P3-memb-SIP (Fig. 5). The high immunogenicity of the P3 idiotype was also revealed in experiments with animals that received a single shot of plasmid DNA (Fig. 6). In this condition, only P3-SIP induced a specific immune response that was already detectable at day 10 and maintained in subsequent extractions. In contrast, 1E10-

Fig. 3. FACS analysis of transfectomas displaying P3-memb-SIP (A) and 1E10-memb-SIP (B) by representative sera from animals immunized by gene gun with P3or 1E10-SIP DNA. Sera were assayed at 1:100 dilution. Filled curve, preimmune serum; thick curve, serum from the animal immunized with the same idiotype as the membrane-displayed one. Reactivity was determined using a FITC-conjugated goat anti-mouse IgG (␥-chain specific) antibody. Arrows indicate the immunogen. Mean fluorescence intensity values are shown in parentheses.

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Fig. 4. IgG response in BALB/c mice immunized by gene gun with SIP DNA corresponding to the H-CDR P3 mutants: R31 → S (A), R98 → S (B), R100a → S (C), and R98;100a → S (D). Sera were obtained 10 days after the third shot and assayed by ELISA at 1:100 dilution against chimeric antibodies and human IgG as indicated. Reactivity was determined using an HRP-conjugated goat anti-mouse IgG (␥-chain specific) antibody.

SIP was not able to induce any antibody response with a single shot of DNA, even at long times post-immunization. Furthermore, the scFv-P3 alone, without any constant region, induced an anti-idiotypic response similar to the one against the P3-SIP (Lopez-Requena et al., 2007). In order to further investigate the nature of the immunogenicity of the P3 idiotype, we tested the anti-idiotypic response in animals from the allogeneic C57BL/6 strain (H2b haplotype) and in athymic BALB/c mice. As shown in Fig. 7, the reactivity against chimeric P3 (Fig. 7A) or 1E10 (Fig. 7B) antibodies was practically absent in the sera from C57BL/6 animals immu-

nized with the respective SIPs. More importantly, a similar result was obtained with the athymic BALB/c mice (Fig. 7C and D). Taken together these results suggest that: (i) a complete immunocompetent T cell compartment is required by P3 to induce the anti-idiotypic antibody response, and (ii) P3 idiotype-derived peptides are specific for BALB/c haplotype (H2d ) and not compatible with the C57BL/6 haplotype (H2b ). The experiments described above were performed using bacterial plasmid DNA, which contains hypomethylated CpG deoxidinucleotides. For this reason we decided to test recombinant adeno-associated virus (rAAV) delivery of the P3-SIP construct, as an alternative way of DNA immunization. Infective rAAV are produced in mammalian cells and therefore, contrary to the bacterial plasmid DNA, the viral DNA does not contain hypomethylated CpGs. As shown in Table 1, the P3-SIP construct delivered by rAAV infection induced an anti-idiotypic specific antibody response in

Fig. 5. FACS analysis on the P3-memb-SIP transfectoma, of sera from animals immunized with the R31 → S-SIP DNA and from animals immunized with the purified chimeric R31 → S antibody. Sera were assayed at 1:100 dilution. Arrows indicate the immunogen in each case. Filled curve, preimmune serum. Reactivity was determined using a FITC-conjugated goat anti-mouse IgG (␥-chain specific) antibody.

Fig. 6. IgG response in BALB/c mice immunized once by gene gun with the P3or 1E10-SIP DNA. Sera were taken 10, 38 or 101 days after the unique shot and assayed by ELISA at 1:100 dilution against chimeric P3 and 1E10 antibodies, as indicated. Columns represent the mean of the results with the three animals immunized in each group. Reactivity was determined using an HRP-conjugated goat anti-mouse IgG (␥-chain specific) antibody.

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Fig. 7. IgG response in C57BL/6 (A and B) or athymic BALB/c (C, D) mice immunized by three shots of gene gun with the SIP DNA: P3 (A and C) or 1E10 (B and D). Sera taken 10 days after the third shot were assayed by ELISA at 1:100 dilution against chimeric P3 (A and C) or 1E10 (B and D) antibodies. Reactivity was determined using an HRP-conjugated goat anti-mouse IgG (␥-chain specific) antibody. The results with sera from immunized BALB/c mice are included as positive controls. Table 1 Summary of the reactivity of sera from BALB/c mice immunized by rAAV-SIP infection against chimeric P3 and 1E10 antibodiesa Immunogen

ch P3 Ab ch 1E10 Ab

P3-SIP

1E10-SIP

3/9 0/9

0/9 0/9

a Results represent number of mice with reactivity (at least twice the preimmune signal) against the chimeric antibodies over total number of immunized mice. Mice were infected with rAAV-SIP particles and sera were taken after a month.

three out of nine animals immunized. In contrast, a similar construct with the 1E10 idiotype was completely unable to induce anti-idiotypic antibodies. These results further strengthen the characteristic immunogenicity of P3 idiotype. 4. Discussion In contrast to other anti-ganglioside IgM mAbs, P3 mAb is highly immunogenic in the syngeneic model (V´azquez et al., 1998). 14F7 mAb, another anti-GM3(NeuGc) ganglioside antibody (Carr et al., 2000), requires coupling to keyhole lympet haemocyanin (KLH) and emulsification in Freund’s adjuvant for eliciting an anti-idiotypic response in BALB/c mice (Rodr´ıguez et al., 2007). P3 mAb is a germ-line IgM (P´erez et al., 2001) while 14F7 is a hypermutated IgG (Rodr´ıguez et al., 2007). Immunogenicity of P3 was also verified with the chimeric version of the molecule, because anti-idiotypic antibodies were the main component of the humoral response in mice immunized with the recombinant antibody (Lopez-Requena et al., 2003, 2007a). The aim of immunizing mice with the SIPs was precisely to provide a stronger evidence of the independence of the iso-

type for the P3 idiotype to elicit an antibody response in the syngeneic model. DNA immunization was used as a convenient and alternative way for delivering the molecule, which in all the previous experiments had been administered as purified protein. The absence of response against the peptide linker of the scFv in the SIP constructs has previously been reported (Cesco-Gaspere et al., 2005). However, not only antibodies against the P3 idiotype were obtained by gene gun immunization, but also against the 1E10 idiotype. It has been previously demonstrated that this idiotype is immunogenic in BALB/c mice only if coupled to a human isotype (Lopez-Requena et al., 2003) or, as 14F7 mAb, when coupled to KLH and emulsified in Freund’s adjuvant (V´azquez et al., 1998). Nevertheless, the anti-idiotypic response was immunodominant not only in the case of mice immunized with the chimeric antibody (Lopez-Requena et al., 2003), but also for the murine antibody administered to xenogeneic models (Alfonso et al., 2002; D´ıaz et al., 2003; Hern´andez et al., 2005). The most surprising finding, in particular for the P3-SIP but also for some animals immunized with the SIPs of 1E10 or the P3 mutants, was the generation upon gene gun immunization of antibodies able to recognize proteins with the human IgG isotype. This response seemed to depend on hyperimmunization, because it was not observed in mice that received only one shot of the P3-SIP-coding plasmid. Antibodies generated by immunization with the P3 H-CDR R → S mutants in SIP format recognized also the wild P3 idiotype. This result in the case of the H-CDR1 R31 → S mutant differs notably from that obtained in mice immunized with the purified chimeric mutant protein (Lopez-Requena et al., 2007a), where the presence of such antibodies could not be demonstrated. As it was also observed that the mutant antibody lost the idiotypic immunodominance or antigenicity of the wild type version, it is still possible that P3 idiotype-specific antibodies could have been

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generated but not detected at the dilution assayed. This explanation is consistent with the fact that antibodies specific for the original idiotype could be detected only in the sera with the highest titers from animals immunized with the H-CDR3 R → S mutants (Lopez-Requena et al., 2007a). Also, the differences in antibody titers observed in animals immunized with the purified mutant chimeric antibodies were practically absent in mice immunized with the corresponding SIP DNA. Bacterial DNA immunization could be related to these unexpected antibody responses. Hypomethylated CpG sequences, typical of bacterial DNA, stimulate cells expressing the Toll-like receptor (TLR) 9 (Hemmi et al., 2000; Wagner, 2004). This interaction initiates an immunomodulatory cascade including the activation of B and T lymphocytes, “natural killer” cells, monocytes, macrophages and dendritic cells (Ashkar and Rosenthal, 2002; Klinman, 2004). However, clear differences in the specificity of the anti-idiotypic antibodies induced with protein as opposed to DNA immunizations have been previously described (Benvenuti and Burrone, 2001). The results with animals inoculated with the SIP-coding rAAV are in agreement with this interpretation. Although only one third of the animals immunized by rAAV infection with the P3-SIP developed an antibody response against this idiotype, no polyspecific reactivity was observed. In none of the animals immunized with the 1E10-SIP using this approach any seroconversion was demonstrated. While plasmid vectors are amplified in bacteria, the recombinant viral particles are produced in mammalian cells, using a system that does not require the helper adenovirus (Matsushita et al., 1998; Grimm and Kleinschmidt, 1999). In contrast with adenovirus, AAV-based vectors do not activate innate (Zaiss et al., 2002; Zaiss and Muruve, 2005) nor adaptive immunity (Bessis et al., 2004). The antibody response against the P3 and 1E10 idiotypes obtained in BALB/c mice by gene gun immunization was not reproduced either in the allogeneic C57BL/6 model or in athymic BALB/c mice. These results strongly suggest the involvement of helper T cells in this response, in view of the MHC-H2 haplotype differences between both strains and the absence of T cells in the athymic animals. This is consistent with previous observations regarding the lack of anti-idiotypic response in these latter mice, when immunized with purified P3 mAb protein without adjuvants or with purified 1E10 mAb protein coupled to KLH and emulsified in Freund’s adjuvant (P´erez et al., 2002). It thus appears that both idiotypes contain peptides capable to associate with MHC class II molecules of the H2d haplotype and initiate a T helper response to assist the anti-idiotypic specific B cells. Although the gene gun immunization could have contributed to an enhanced efficiency of the anti-idiotypic response by activating TLR in antigen-presenting cells, as observed for the anti-1E10 idiotype response, the intrinsic immunogenicity of the P3 idiotype was further confirmed when expressed from the rAAV infective particles, which do not contain activating CpGs and do not infect efficiently antigen-presenting cells (Bessis et al., 2004). It would be interesting to determine the frequency of idiotypes with the unusual properties of P3 in the repertoire of neonatal mice as well as in healthy or autoimmune adults.

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