insights into the immunogenetic basis of two ganglioside-associated idiotypic networks

ARTICLE IN PRESS

Immunobiology 212 (2007) 57–70

0171-2985/$ - se

doi:10.1016/j.im

Abbreviations

enzyme-linked i

layer chromato

acetyl; NeuGc,

variable heavy�CorrespondE-mail addr

1Both author

www.elsevier.de/imbio

Insights into the immunogenetic basis of two ganglioside-associated

idiotypic networks

Mabel Rodrıguez1, Lourdes Roque-Navarro1, Alejandro Lopez-Requena,Ernesto Moreno, Cristina Mateo de Acosta, Rolando Perez, Ana Marıa Vazquez�

Department of Antibody Engineering, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba

Received 17 July 2006; received in revised form 4 August 2006; accepted 14 August 2006

Abstract

The heavy-chain variable regions (VH) from 14F7 MAb, an IgG1 antibody specific for GM3(NeuGc) ganglioside,and its anti-idiotype, the 4G9 MAb, were cloned and sequenced. Comparison with previously reported sequencesshowed that VH 14F7 belongs to the J558(VHI) gene family and that it is highly mutated. VH 4G9 belongs to theQ52(VHII) gene family. The HCDR3 14F7 sequence contains three basic residues that could be involved in the bindingto 4G9 MAb, which bears acidic residues in its HCDR3. Studies performed in the syngeneic model showed that 14F7MAb requires both coupling to KLH and the use of Freund’s adjuvant to induce an effective anti-idiotypic IgG (Ab2)response. In contrast, P3 MAb, a germline gene-encoded Ab1 that also recognizes the GM3(NeuGc) gangliosidethrough a basic motif in its H-CDRs, has been reported to be immunogenic in syngeneic mice, even when injected insaline. In addition, when Leghorn chickens were immunized with 14F7 or P3 MAbs emulsified in Freund0s adjuvant,only P3-immunized animals were able to develop antibodies that recognized NeuGc-containing gangliosides, antigenswhich are not present in the normal tissues of this animal species. This phenomenon could be due to the lack ofidiotypic connectivity of 14F7MAb.r 2006 Elsevier GmbH. All rights reserved.

Keywords: Ganglioside; Idiotype; Immunogenetic; MAb; Idiotypic network; Neugc-containing ganglioside

Introduction

Gangliosides are sialic acid-containing glycosphingo-lipids normally expressed in the plasma membrane of

e front matter r 2006 Elsevier GmbH. All rights reserved.

bio.2006.08.005

: CDR, complementarity determining region; ELISA,

mmunosorbent assay; HPTLC, high-performance thin

graphy; MAb, monoclonal antibody; NeuAc, Neu-

Neu-glycolyl; PCR, polymerase chain reaction; VH,

chain; VL, variable light chain

ing author. Tel.: +537 2716810; fax: +53 7 2720644.

ess: [email protected] (A. Marıa Vazquez).

s equally contributed to this work.

eukaryotic cells (Wiegandt, 1985), but their expressionpatterns change during oncogenic transformation(Hakomori, 1985; Irie and Ravindranath, 1990). TheN-glycolyl (NeuGc)-neuraminic acid variant of ganglio-sides is widely expressed in most mammalian tissues, butis rarely found in normal human cells. However, thepresence of NeuGc-sialic acid has been reported inhuman tumors (Higashi et al., 1984, 1988; Hirabagashiet al., 1987; Miyake et al., 1990; Devine et al., 1991;Watarai et al., 1995; Marquina et al., 1996; Malykhet al., 2001). Chickens are another vertebrate specieswhere NeuGc-glycoconjugates are not expressed innormal tissues and therefore a strong antibody response

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dx.doi.org/10.1016/j.imbio.2006.08.005

mailto:[email protected]

ARTICLE IN PRESSM. Rodrıguez et al. / Immunobiology 212 (2007) 57–7058

against this sialic acid variant can be obtained inimmunized animals (Fujii et al., 1982; Ledeen and Yu,1982).

A murine IgM monoclonal antibody, named P3, wasgenerated in our group by immunizing BALB/c micewith liposomes containing GM3(NeuGc) gangliosideand tetanus toxoid. This MAb reacts with a broadbattery of N-glycolyl-containing gangliosides and sul-fated glycolipids (Vazquez et al., 1995a; Moreno et al.,1998). Later we reported the generation of 14F7 MAb, ahighly specific IgG1 antibody against GM3(NeuGc)ganglioside that was produced by immunizing BALB/cmice with this ganglioside hydrophobically conjugatedto human very low density lipoproteins (VLDL) (Carret al., 2000). Both MAbs were able to react with antigensexpressed in human melanoma and breast tumors(Vazquez et al., 1995a; Carr et al., 2000; Alfonsoet al., 2002; Oliva et al., 2006). P3 and 14F7 MAbs,coupled to a carrier protein and in the presence ofFreund’s adjuvant, induced anti-idiotypic responses insyngeneic mice, and specific Ab2 MAbs were generatedand characterized (Vazquez et al., 1998; Rodrıguezet al., 2003). In addition, P3 MAb has the uncommonability to induce an IgG anti-idiotypic antibodyresponse in BALB/c mice even when administered insaline (Vazquez et al., 1998). On the other hand, 1E10(anti-P3) and 4G9 (anti-14F7) Ab2 MAbs were not ableto induce anti-GM3(NeuGc) ganglioside Ab3 antibodiesin syngeneic model, but induced an antigen-positivehumoral response in chickens (Rodrıguez et al., 2003;Hernandez et al., 2005) and, in the case of 1E10 MAb,also in humans (Alfonso et al., 2002; Dıaz et al., 2003),where N-glycolyl-gangliosides are non-self-antigens.

P3 is a member of the Q52 VH family, which has beenassociated with high idiotypic connectivity (Holmberg,1987). Although some authors claim that somatichypermutations can be responsible of increasing theimmunogenicity of antibodies, P3 MAb is germlinegene-encoded (Perez et al., 2001). It has also beenpostulated that the immunoglobulin isotype is crucialfor the immunogenic properties (Reitan and Hannestad,1995). However, we previously showed that, at least inthe case of P3 MAb, this is not a satisfactoryexplanation for its high immunogenicity in the syngenicmodel (Vazquez et al., 1998; Lopez-Requena et al.,2003). Structural modelling as well as immunogeneticanalysis of P3 MAb variable regions suggested theinvolvement of particular basic residues of the H-CDRsin the biological properties of this antibody. Theimportance of these residues for both its bindingproperties and its immunogenicity has indeed beendemonstrated (Lopez-Requena et al., 2007a).

Taking into account all those previous data relatedwith P3 MAb, we decided to analyze if we could findsome relationship between P3 MAb and 14F7 MAbidiotypic networks. Also, we look for a correlation of the

immunogenic properties of these Ab1 antibodies withtheir immunogenetic features. We sequenced and ana-lyzed the variable regions of Ab1 14F7 MAb and Ab24G9 MAb, comparing them with the reported sequencesof Ab1 P3 MAb and its anti-idiotypic MAbs (Perez et al.,2001), respectively. While P3 and 1E10 MAbs are ofgermline origin both 14F7 and 4G9 MAbs are hypermu-tated antibodies and belong to different VH and VL genefamilies. Although the two Ab1 and Ab2 share somemotifs of charged amino acids in their respective H-CDR3, no shared idiotopes could be demonstrated. P3MAb was able to induce an anti-N-glycolyl gangliosideantibody response in chickens, while 14F7 MAb was not.This result, together with the lack of immunogenicity of14F7 MAb in the syngeneic model when administeredwithout carrier protein, seem to indicate that an antibodyresulting from a mature response is less idiotypicallyconnected than a germline antibody.

Materials and methods

Animals

Six to eight week old female BALB/c mice andLeghorn chickens (10-week old) were purchased fromthe Center for Laboratory Animal Production (CEN-PALAB, Havana, Cuba). Animal care was in accor-dance with our institutional guidelines.

Gangliosides

GM3(NeuGc) was isolated from horse erythrocytes(Stults et al., 1989); GM2(NeuGc) was prepared frommouse BALB/c liver (Hashimoto et al., 1983);GM3(NeuAc) was isolated from sheep spleens (Sven-nerholm, 1976) and GM2(NeuAc) was purchased fromSigma (St. Louis, MO).

MAbs and their fragments

P3 MAb (IgM, k) was purified from ascitic fluid by gelfiltration chromatography using a Sephacryl S-300 high-resolution column (Pharmacia, Uppsala, Sweden) equi-librated with phosphate-buffered saline (PBS) contain-ing 0.5M NaCl. 14F7 MAb (IgG1, k) was purified byProtein-A affinity chromatography (Pharmacia) andanalyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) under reducing condi-tions. Syngeneic 1E10, 3F9 and 3B11, and 4G9 Ab2MAbs (IgG1, k), specific to P3 and 14F7 Ab1 MAbs,respectively (Vazquez et al., 1998; Rodrıguez et al., 2003;Perez et al., 2001; Lopez-Requena et al., 2007b), werepurified as mentioned above. The following murineMAbs were used as isotype control: E1 MAb (IgM, k)

ARTICLE IN PRESSM. Rodrıguez et al. / Immunobiology 212 (2007) 57–70 59

against GM2 ganglioside (Alfonso et al., 1995), ior-c5MAb (IgG1, k, anti-colorectal antigen) (Vazquez et al.,1995b) and ior-CEA-1 MAb (IgG1, k) against thecarcinoembryonic antigen (Tormo et al., 1989). Chi-meric P3 (Lopez-Requena et al., 2003) and chimeric14F7 (our unpublished data) antibodies bear therespective murine variable regions and the human g1constant region. F(ab0)2 fragments from 14F7, 4G9,1E10, ior-CEA-1 and ior-c5 MAbs were obtained usinga standard procedure previously described (Coliganet al., 1995).

cDNA cloning and sequencing of heavy- and light-

chain variable regions of 14F7 and 4G9 MAbs

Total RNA was extracted with the Trizol reagent(Gibco-BRL, Paisley, Scotland). First strand cDNAsynthesis and PCR amplification of V-genes wereperformed using the Access RT-PCR kit, as describedby the manufacturer (Promega, Madison, USA). In thecase of the light-chain variable region, the DNAfragment was amplified using specific primers as follows:50-GGG GAT ATC CAC CAT GGT (AT)T (TC)C(TA)C ACC TCA G(AT) T(AC) CTT GGA CTT-30

and 50-AGC GTC GAC TTA CGT TTC AGC TCCAGC TTG GTC CC-30 for 14F7, and 50-GTA CTCCAG TCG ACG ATA TCC AGA TGA C(AC) CA(GA)AC T(AC) C-30 and 50-AAG GAA AAA AGC GGCCGC TTT (TC)A (TG)(T C)T CCA GCT TGG T-30 for4G9. Heavy-chain variable region was amplified usingthe following specific primers: 50-GGG GAT ATC CACCAT GGA AAG GCA CTG GAT CTT TCT CTTCCT G-30 and 50-GGG GCT AGC TGA GGA GACGGT GAC CGT GGT-30 for 14F7, and 50-GGG GATATC CAC CAT GGC TGT CTT GGG GCT GCTCTT CT-30 and 50-GGG GCT AGC TGC AGA GACAGT GAC CAG AGT-30 for 4G9. Purified productswere cloned into pMOSBlue vector (Amersham Phar-macia Biotech, UK) for sequencing of variable regionsusing the T7 Sequencing kit (Amersham PharmaciaBiotech), as described by the manufacturer.

Assignment of the origin of the variable gene

segments utilized

The origin of the variable gene segments was assignedas previously described (Perez et al., 2001). In brief,individual VH and VL sequences were searched againstthe IMGT database for sequence similarity with knownmurine germline genes, as possible donor sequence ofthe rearranged gene. The JK and JH regions sequencedwere compared with Kabat database genes (Kabat et al.,1991). The analysis of the junctional sequences in VHsequences was performed as described by Bangs et al.(1991). D region nucleotides were identified after

alignment of heavy-chain CDR3 nucleotide sequenceswith known murine D minigenes (Kabat et al., 1991).When junctional nucleotides could have been contrib-uted by either D or the adjacent VH or JH segment, theywere assigned to VH or JH. In cases where no VH or JHnucleotides were deleted during recombination with theD gene, possible P additions were identified (Robbinsand Nisonoff, 1987). N additions were identified whereno D, VH, JH or P nucleotides could be assigned.

Induction of antibody response against 14F7 and P3

MAbs in syngeneic and xenogeneic animals

To study the capacity of P3 and 14F7 MAbs to induceAb2 antibody-producing cells, groups of seven BALB/cmice were immunized subcutaneously with 100 mg of P3or 14F7 MAbs emulsified in complete Freund’s adju-vant, and a week later mice received a booster injectionof 50 mg of the MAbs emulsified in incomplete Freund’sadjuvant.

To evaluate serological Ab2 response, groups of sevenBALB/c mice were immunized subcutaneously, at14-day intervals, with three doses of 50 mg of Ab1MAbs, either free or coupled to keyhole limpethemocyanin (KLH) as previously reported (Raychaud-huri et al., 1986), and emulsified in complete Freund’sadjuvant for the first injection and in incompleteFreund’s adjuvant in subsequent doses. Animal serumsamples were collected before starting the protocol and 7days after the final dose.

Groups of three chickens were immunized with fourdoses of 100 mg of 14F7 or P3 MAbs emulsified inFreund’s adjuvant at 2-week intervals, and serumsamples were obtained before starting immunizationsand 1 week after the animals received the last dose.Ior-c5 MAb was used as control.

Antibody binding assays

To measure the presence of anti-idiotypic antibodiesin the sera of P3- and 14F7-immunized mice, a solidphase ELISA was performed as previously described(Vazquez et al., 1998). Briefly, Maxisorp 96-wellmicrotiter plates (Nunc, USA) were coated overnightat 4 1C with 10 mg/mL of P3 MAb and F(ab0)2 fragmentsof 14F7 MAb in carbonate buffer (pH 8.9). E1 MAband F(ab0)2 fragments of ior-CEA1 MAb were used asisotype-matched controls. After washing with PBScontaining 0.05% Tween 20, the plates were blockedfor 1 h at room temperature with PBS containing 0.05%Tween 20 and 1% bovine serum albumin (BSA).Different serum dilutions from mice immunized withP3 and 14F7 MAbs were then added to the plates andincubated for 2 h at 37 1C. After washing, alkalinephosphatase-conjugated goat anti-mouse IgG or IgM,


(Fcg or Fcm specific, Jackson, West Grove, PA) wereadded and incubated for 1 h at 37 1C. The plates werethen washed and the reaction was developed withp-nitrophenylphosphate substrate (Sigma) in diethano-lamine buffer, pH 9.8. Absorbance was measuredat 405 nm in an ELISA reader (Organon Teknike,Salzburg, Austria).

Binding of chicken sera to Ab1 MAbs was measuredusing the ELISA assay described above, where the plateswere coated with P3 or 14F7 MAbs. Alkaline phospha-tase conjugated-rabbit anti-chicken IgY (Sigma) wasused as second antibody.

Antibody binding inhibition assay

In order to determine the anti-idiotype component inthe antibody response in chickens immunized with 14F7and P3 MAbs, different animal serum dilutions wereincubated overnight at 4 1C with 500 mg/mL (finalconcentration) of isotype-matched irrelevant MAb.These mixtures were added to ELISA microplatescoated with Ab1 MAbs or the irrelevant antibody used.After incubation for 2 h at 37 1C, the plates were washedand alkaline phosphatase conjugated-rabbit anti-chick-en IgY (Sigma) were added as described above.

Inhibition of Ab1-Ab2 binding by Ab3 sera wasmeasured by ELISA with chimeric Ab1s bound tomicrotiter plates. Hyperimmune chicken sera wereadded and incubated 2 h at 37 1C. After washing theplates, anti-idiotypic Ab2 MAbs (4G9 for 14F7 MAb,and 1E10, 3F9 and 3B11 MAbs for P3 MAb) wereadded and incubated 1 h at 37 1C. Plates were washedand bound Ab2 MAbs were detected by alkalinephosphate conjugated anti-mouse IgG Fcg-specific(Jackson). Percent inhibition of Ab2 MAb binding wascalculated relative to the binding of these MAbs in theabsence of inhibitor.

Anti-ganglioside response induced in xenogeneic

model

Binding of chicken sera to purified gangliosides wasdetermined using an ELISA assay described previously(Rodrıguez et al., 2003). Briefly, GM3(NeuGc), GM3(NeuAc), GM2(NeuGc) and GM2(NeuAc) (4 mg/mL) in50 ml of methanol were dried in 96-well microtiter plates(Polysorp, Nunc). Plates were blocked with 1% BSA in0.05M Tris–HCl buffer, pH 7.8, for 30min at 37 1C.Serum samples were incubated 2 h at 37 1C, and afterwashing with PBS, alkaline-phosphatase-conjugatedrabbit anti-chicken IgY (Sigma) were added to theplates and incubated for 1 h at 37 1C. The wells werewashed again and substrate solution was added.

The presence in chicken sera of antibodies specific togangliosides was also detected by immunostaining on

HPTLC plates, as described previously (Kawashima etal., 1993; Hernandez et al., 2005). Briefly, HPTLC plates(Merck, Darmstadt, Germany) were used for theglycolipid fractionation using chloroform/methanol/0.2% CaCl2 in 2.5M NH3 (5:4:1, v:v:v). TLC plateswere stained with the orcinol reagent. Then, the plateswere soaked in hexane containing 0.1% poly-isobutyl-methacrylate (Aldrich Chemical Company Inc., Mil-waukee, WI) and were incubated with serum dilutionsfor 2 h at room temperature. After washing with PBS,the plates were incubated with biotin-SP-conjugatedanti-chicken IgY (IgG) (Jackson), followed by strepta-vidin-conjugated peroxidase for 1 h at room tempera-ture. The plates were washed again and incubated withthe substrate solution consisting of 40 mg/mL o-pheny-lendiamine (Sigma) in 80mM citrate-phosphatasebuffer, pH 5.0, containing 0.12% H2O2.

Results

Analysis of VH and VL gene usage by the 14F7 and

4G9 MAbs

The nucleotide and the deduced amino acid sequencesof the heavy- and light-chain variable regions of 14F7and its anti-idiotype 4G9 MAbs are shown in Figs. 1and 2, respectively. The GenBank accession numbersAY331718, AY331717, AY682092 and DQ020030correspond to VH 14F7, Vk 14F7, VH 4G9 and Vk4G9, respectively.

The 14F7 MAb

When compared with germline VH segments, thecDNA sequence of VH 14F7 showed 91% nucleotideidentity from codons 1 to 94 with the VH region ofH280-15 MAb (GenBank accession no. M36225), amember of the J558 (VHI) gene family (Kavaler et al.,1990). Both sequences differ in 25 nucleotide positions,with CDR2 and FRs 1 and 3 being the most divergent(Fig. 1A). The sequence most similar to VH 14F7 is thecDNA sequence of JL1/13-8 MAb (GenBank accessionno. AF316525), generated in BALB/c mice immunizedwith 2-phenyl-oxazolone (Lange et al., 1999), belongingto the same family as VH 14F7. Both sequences share a92% nucleotide identity (codons 1–94) with differencesin 23 nucleotides, which resulted in 17 divergent aminoacid positions, FR3 being the most divergent region.Differences observed between VH 14F7 and the nucleo-tide sequences of VH JL1/13-8 and VH H280-15 arecoincident in type and distribution, except for oneconservative change in FR3 (Leu89-Val89) in VH 14F7with respect to VH H280-15. The JH gene segment usedby 14F7 MAb is JH4.

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(A)

(B)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Q V Q L Q Q S G N E L A K P G A S M K M S C R A

VH 14F7 CAG GTC CAG CTT CAG CAG TCT GGG AAT GAA CTG GCA AAA CCT GGG GCC TCA ATG AAG ATG TCC TGC AGG GCT

A V K

H280-15 --- --- --- --- --- --- --- --- GC- --- --- --- --- --- --- --- --- G-- --- --- --- --- -A- ---

_______CDR1_______

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

S G Y S F T S Y W I H W L K Q R P D Q G L E W I

VH 14F7 TCT GGC TAC TCC TTT ACT AGC TAC TGG ATA CAC TGG TTA AAA CAG AGA CCT GAC CAG GGT CTG GAA TGG ATT

T M V G

H280-15 --- --- --- A-- --- --- --- --- --- --G --- --- G-- --- --- --G --- -GA --- --- --- --- --- ---

________________________________CDR2______________________________

49 50 51 52 52a 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71

G Y I D P A T A Y T E S N Q K F K D K A I L T A

VH 14F7 GGA TAC ATT GAT CCT GCC ACT GCT TAT ACT GAG TCC AAT CAG AAG TTC AAG GAC AAG GCC ATA TTG ACT GCA

N S G Y T

H280-15 --- --- --- A-- --- AG- --- -G- --- --- --- -A- --- --- --- --- --- --- --- --- -C- --- --- ---

72 73 74 75 76 77 78 79 80 81 82 82a 82b 82c 83 84 85 86 87 88 89 90 91 92

D R S S N T A F M Y L N S L T S E D S A V Y Y C

VH 14F7 GAC AGA TCC TCC AAC ACA GCC TTC ATG TAT CTG AAC AGC CTG ACA TCT GAA GAC TCT GCA GTC TAT TAC TGT

K S Y Q S L

H280-15 --- -A- --- --- -G- --- --- -A- --- C-A --- -G- --- --- --- --- --G --- --- --- C-G --- --- ---

________________________CDR3____________________________________

93 94 95 96 97 98 99 100 a b c d e f g h 101 102 103 104 105 106 107 A R E S P R L R R G I Y Y Y A M D Y W G Q G T

VH 14F7 GCA AGA GAG TCT CCT A GG TTA CGA CGC GGT ATA TAT TAC TAT GCC ATG GAC TAC TGG GGT CAA GGA ACC

H280-15 --- ---

T

DSP2.3,4 -- A-- -T-- --- --- -

DSP2.3,4,6 -- --- --- -

JH4 --- --- --T --- --- --- ---

___

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

D L V L T Q S P A T L S V T P G D S V S F S C R

Vκ 14F7 GAT CTT GTG CTA ACT CAG TCT CCA GCC ACC CTG TCT GTG ACT CCA GGA GAT AGC GTC AGT TTT TCC TGC AGG

I L

23-43 --- A-- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- C-- --- --- ---

_______________CDR1___________________

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

A S Q S I S N N L H W Y Q Q R T H E S P R L L I

Vκ 14F7 GCC AGC CAA AGT ATT AGC AAC AAC CTA CAC TGG TAT CAA CAA AGA ACA CAT GAG TCT CCA AGG CTT CTC ATC

K S

23-43 --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- T-- --- --- --- --- --- --- --- ---

___________CDR2___________

49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72

Y K A S Q S I S G I P S R F S G S G S G T D F T

Vκ 14F7 AAG TAT GCT TCC CAG TCC ATT TCT GGG ATC CCC TCC AGG TTC AGT GGC AGT GGA TCA GGG ACA GAT TTC ACT

23-43 --- --- --- --- --- --- --C --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

________________CDR3__________

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

L S I I S V E T E D F G M Y F C Q R S N R W P L

Vκ 14F7 CTC AGT ATC ATC AGT GTG GAG ACT GAA GAT TTT GGA ATG TAT TTC TGT CAG CAG AGT AAC AGG TGG CCT CTC

N S

23-43 --- --- --- -A- --- --- --- --- --- --- --- --- --- --- --- --- --A --- --- --- --C --- ---

__

97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119

T F G A G T K L E L K R A E A A P T V S S S S

Vκ 14F7 ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA CGG GCT GAG GCT GCA CCA ACT GTA TCA TCT TCA AGC

Fig. 1. Nucleotide and deduced amino acid sequences of the cDNA encoding the heavy (VH 14F7) and light (VK 14F7) chain

variable region of 14F7 MAb. Amino acids are numbered according to Kabat et al. (1991). Dashed lines indicate nucleotide

sequence identities. The amino acid residue encoded by each codon is given above the nucleotide sequence: (A) VH 14F7 compared

with the germline sequence H280-15 (Schaeble et al., 1999), including the possible origin of CDR3H. (B) VK 14F7 compared with

the germline sequence 23-43 (Kavaler et al., 1990). Dashed lines indicate homology with known D and J genes. N nucleotides are

represented in bold italics and P nucleotides are underlined. The germline sequences of DSP2.2, DSP2.3, DSP2.4 and DSP2.6,

together with JH4 sequences are from Kabat et al. (1991).

M. Rodrıguez et al. / Immunobiology 212 (2007) 57–70 61

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Q V Q L K E S G P G L V A P S Q S L S I T C T V

VH 4G9 CAG GTG CAG CTG AAG GAG TCA GGA CCT GGC CTG GTG GCG CCC TCA CAG AGC CTG TCC ATC ACT TGC ACT GTC

VH Ox-1 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

_______CDR1_______

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

S G F S L S N Y G V H W V R Q P P G K G L E W L

VH 4G9 TCT GGG TTT TCA TTA TCC AAC TAT GGT GTA CAC TGG GTT CGC CAG CCT CCA GGA AAG GGT CTG GAG TGG CTG

T S

VH Ox-1 --- --- --- --- --- A-- -G- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

_____________________________CDR2_____________________________

49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72

G E I W A G G S T N Y N S A L M S R L S I S K D

VH 4G9 GGA GAA ATA TGG GCT GGT GGA AGC ACA AAT TAT AAT TCG GCT CTC ATG TCC AGA CTG AGC ATC AGC AAA GAC

V

VH Ox-1 --- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

73 74 75 76 77 78 79 80 81 82 a b c 83 84 85 86 87 88 89 90 91 92 93

N S K S Q V F L K M N S L Q T D D T A M Y Y C A

VH 4G9 AAC TCC AAG AGC CAA GTT TTC TTA AAA ATG AAC AGT CTG CAA ACT GAT GAC ACA GCC ATG TAC TAC TGT GCC

VH Ox-1 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

________________CDR3______________________

94 95 96 97 98 99 100 a b c 101 102 103 104 105 106 107 108 109 110 111 112 113

R A Y D Y D G A W F A Y W G Q G T L V T V S A VH 4G9 AGA GCG TAT GAT TAC GAC GGG GCC TGG TTT GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC TCT GCA

VH Ox-1 ---

DSP2.2 --- --- --- ---

JH3 --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

__

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

D I Q M T Q T T S S L S A S L G D R V T I S C R

Vκ 4G9 GAT ATC CAG ATG ACA CAG ACT ACA TCC TCC CTG TCT GCC TCT CTG GGA GAC AGA GTC ACC ATC AGT TGC AGG

91A3 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

_____________CDR1_____________________

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

A S Q D I S N Y L N W Y Q Q K P D G T V K L L I

Vκ 4G9 GCA AGT CAG GAC ATT AGC AAT TAT TTA AAC TGG TAT CAG CAG AAA CCA GAT GGA ACT GTT AAA CTC CTG ATC

91A3 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

__________ CDR2___________

49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72

Y Y T S R L H S G V P S R F S G S G S G T D Y S

Vκ 4G9 TAC TAC ACA TCA AGA TTA CAC TCA GGA GTC CCA TCA AGG TTC AGT GGC AGT GGG TCT GGA ACA GAT TAT TCT

91A3 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

_______________CDR3____________

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

L T I S N L E Q E D I A T Y F C Q Q G D T L P Y

Vκ 4G9 CTC ACC ATT AGC AAC CTG GAG CAA GAA GAT ATT GCC ACT TAC TTT TGC CAA CAG GGT GAT ACG CTT CCG TAC

N

91A3 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- A-- --- --- --T C

__

97 98 99 100 101 102 103 104 105

T F G G G T K L E

Vκ 4G9 ACG TTC GGA GGG GGG ACC AAG CTG GAA

Fig. 2. Nucleotide and deduced amino acid sequence of the cDNA encoding the heavy (VH 4G9) and light (VK 4G9) chain variable

region of the anti-idiotypic 4G9 MAb: (A) VH 4G9 compared with the germline sequence, VHOx-1 gene (Kaartinen et al., 1989). (B)

VK 4G9 compared with the germline sequence, 91A3 gene (Sanz and Capra, 1987). (For details, see legend of Fig. 1.)

M. Rodrıguez et al. / Immunobiology 212 (2007) 57–7062

The VL sequence of 14F7 MAb belongs to the VK23family, and it is 97% identical from codons 1 to 95 tothe germline gene 23–43 (GenBank accession no.AJ235973) (Schaeble et al., 1999) (Fig. 1B). Thesequence most related to Vk 14F7 reported in theGenBank database (accession no. M16162) is the cDNAsequence of MAK33 MAb (96% nucleotide identity),generated in BALB/c mice against MM creatine kinase

(Buckel et al., 1987). The Vk segment sequence of 14F7MAb differs from 23 to 43 gene in eight nucleotides,which originates differences in five amino acids, locatedin FR1 (Ile2-Leu2, Leu21-Phe21), FR2 (Lys39-Arg39, Ser40-Thr40) and in FR3 (Asn76-Ile76). Theremaining differences correspond to silent mutations.The FR4 of the light chain of 14F7 MAb is coded by theJk5 gene.

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Table 1. Distribution and classification of mutations in VH

and VK regions of 14F7 MAb, and VH region of P3 MAb,

when compared with the most homologous germline sequences

reported in Genbank or IMGT databases

Regions M S R R/S

C NC

VH 14F7a

FR FR1 4 0 3 1 6

FR2 3 1 1 1

FR3 7 1 1 5

CDR CDR 1 1 0 1 0 4/0

CDR 2 4 0 2 2

Vk 14F7b

FR FR1 2 0 1 1 5/0

FR2 2 0 2 0

FR3 1 0 0 1

CDR CDR 1 0 0 0 0 1/2

CDR 2 1 1 0 0

CDR3 2 1 0 1

VH P3c

FR FR1 0 0 0 0 0/1

FR2 1 1 0 0

FR3 0 0 0 0

CDR CDR 1 0 0 0 0 1/0

CDR 2 1 0 1 0

M: total of mutations, S: silent mutations, R: replacements, C:

conservative changes, NC: non-conservative changes.aComparison between VH 14F7 and VH JL1/13-8 sequence (Lange

et al., 1999).bComparison between Vk 14F7 and Vk gene 23-43 (Schaeble et al.,

1999).cComparison between VH P3 and VH gene asws1 (Monestier et al.,

1994; Perez et al., 2001).


Table 1 shows the distribution and classification ofpossible mutations existing in VH and VK regions of14F7 MAb when compared with the most similargermline sequences reported in IMGT databases.A quantitative and qualitative analysis of replacement(R) and silent (S) mutations in VH 14F7 showed apattern which is characteristic for hypermutated anti-bodies. The ratio R/S in framework regions of VH 14F7was 6 (12/2), a value significantly greater than 2.9, whichhas been proposed to indicate a positive selection ofnucleotide changes that affect the amino acid sequenceof antibodies (Shlomchik et al., 1990). In heavy-chainCDRs, there is a complete absence of silent mutations.In contrast, Vk 14F7 carries very few amino acidreplacements and, interestingly, only one amino acidchange was observed in the light-chain CDRs. From thefive amino acid replacements that were observed in theFR regions of the light chain of 14F7 MAb, only twoare non-conservative changes.

The 14F7-specific anti-idiotypic 4G9 MAb

VH 4G9 is 97% identical in nucleotide sequence to amember of the Q52 gene family, the germline geneVHOx-1 (Kaartinen et al., 1989) (GenBank accession no.U53526). There are three nucleotide differences betweenVH4G9 and VHOx-1 (VH from codons 1 to 94), leadingto three amino acid replacements, which are distributedin FR1 and CDRs 1 and 2 (Fig. 2A). Two of thesechanges are conservative: (Thr30-Ser30) and (Ser31-Asn31), and only one of the mutations induced a non-conservative amino acid change (Val50-Glu50). The JHgene segment used by 4G9 MAb is JH3. When wecompared VH 4G9 with the sequences reported in theGenBank Database, the 15 most similar sequences toVH 4G9 were also coded by the VHOx-1 gene in itsgermline form, and differ from VH 4G9 in the samepositions as VHOx-1.

Vk4G9 belongs to the gene family Vk10, and shares a98% nucleotide identity with the germline gene 91A3(GenBank accession no. M15520) (Sanz and Capra,1987). The changes observed in the nucleotide sequencein both antibodies lead only to an amino acidreplacement at position 92 (Asn92-Asp92). The othermutation at position 95 is silent (Fig. 2B). The J segmentused is Jk2.

Immunogenetic origin of the HCDR3 of 14F7 MAb

The immunogenetic origin of HCDR3 of 14F7 MAbis shown in Fig. 1A. The HCDR3 segment of 14F7 MAbis coded by 48 nucleotides, 16 of which belong to the Dsegment and 18 to the JH segment. This unusualHCDR3 segment (no similar sequence was found inthe GeneBank) is very large to consider its origin onlyby nucleotide additions from one single gene D. A morelikely explanation is the combination of two minigenesfrom the DSP2 family. Either DSP2.4 or DSP2.3 genes,used in reading frame 2, may have contributed to a firstregion, until the first nucleotide of codon 98. The secondsegment, may have been originated either from DSP2.4,DSP2.3 or DSP2.6 minigenes, used in reading frame 3(changing an adenine by a cytosine in codon 97). Thus,the deletion of a thymine in codon 98 seems to takeplace as the result of the two D minigenes fusion, and anarginine appears at this position. As a consequence, theamino acid sequence Arg98-X99–Arg100–Arg100a is in-troduced in HCDR3 of 14F7 MAb, similar to asequence segment reported for HCDR3 of P3 MAb. Nnucleotides appear at both sides of the D segment, andconstitute only 15% of the sequence of this HCDR3differing from HCDR3 of P3 MAb, in which the Nnucleotides represent almost 50% of HCDR3 (Perezet al., 2001). The region between codons 100e and codon113 belongs to the JH4 gene, with the presence of a silent

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P3 E1 14F7 CEA10.0

0.5

1.0

1.5

2.0

2.5

3.0B

OD

405

nm

Coating MAbs

P3 E1 14F7 CEA1Coating MAbs

0.0

0.5

1.0

1.5

2.0

2.5

3.0A

OD

405

nm

Fig. 3. Anti-idiotypic (Ab2) response induced in BALB/c mice

immunized with 14F7 and P3 MAbs. Sera from representative

BALB/c mice immunized with 14F7 MAb coupled to KLH

and emulsified with Freund’s adjuvant (A) or with 14F7 or P3

MAbs in the presence of adjuvant (B) were diluted 1:1000 and

assayed against F(ab0)2 fragments of 14F7 MAb, purified P3

MAb or isotype-matched control MAbs-coated microtiter

plates. Binding was assessed using alkaline phosphatase-

conjugated goat anti-mouse IgG (Fcg-specific). Gray bars

represent the binding to the different MAbs of the serum from

a mouse immunized with 14F7 MAb, and black bars those

from a mouse immunized with P3 MAb.


mutation (cytosine by thiamine) in codon 100g. The lasttwo nucleotides of codon 100d belong to the 50 extremeof JH4 gene, which may have been conserved during D-JH recombination (P nucleotides) (Lafaille et al., 1989).

Antibody responses generated in the syngeneic model

by 14F7 and P3 MAbs

To compare the immunogenicity of 14F7 and P3MAbs in the syngeneic model, the sera of BALB/c miceimmunized with different doses were tested separatelyby ELISA. A highly specific IgG Ab2 response wasinduced in all animals tested by 14F7 MAb when it wasadministered coupled to KLH and emulsified inFreund’s adjuvant (Fig. 3A). Neither IgM (data notshown) nor IgG Ab2 response were detected in miceimmunized with this Ab1 when it was injected only inthe presence of the adjuvant, at the lowest serumdilution tested (1:100). In contrast, as it has beenpreviously reported by our group (Vazquez et al., 1998),the immunization of BALB/c mice with P3 MAbemulsified in Freund’s adjuvant, induced an evidentIgG Ab2 response that was specific against this MAb,since no binding of sera was detected against 14F7 MAbor other MAbs used as controls, even at the lowestdilution tested (1:100) (Fig. 3B). Furthermore, Ab2-producing B cells (IgG isotype and specific for P3idiotopes) were detected in proximal lymph-nodes frommice immunized with P3 MAb, in agreement with aprevious report (Perez et al., 2002), but not detectable inthe lymph node of the animals immunized with 14F7emulsified in Freund’s adyuvant (data not shown).

Antibody responses in chickens immunized with P3

and 14F7 MAbs

The antibody response induced in chickens byimmunization with P3 or 14F7 MAbs was alsocharacterized. Chickens that received four doses of100 mg of P3, 14F7 or the control antibody ior-c5 in thepresence of adjuvant, generated strong serological anti-body responses against the whole murine MAb moleculeused in each group as immunogen (titer 1:1,000,000),with the exception of one chicken from the groupimmunized with P3 MAb (data not shown).

To analyze the anti-idiotype response against Ab1MAb, hyperimmune animal sera were pre-absorbedwith an isotype-matched control MAb to block the anti-isotype component of the sera. The remnant reactivityagainst P3 and 14F7 MAbs in the pre-absorbed sera wassignificantly higher than the reactivity of the absorbedsera against the control MAb (Fig. 4).

Hyperimmune sera from two of three chickensimmunized with P3 MAb specifically inhibit the bindingof 1E10, 3F9 and 3B11 MAbs (Ab2) to P3 MAb (Ab1)

(Fig. 5). A similar result was obtained in all 14F7-immunized chickens, since 4G9 MAb (Ab2) binding toAb1 MAb was inhibited by hyperimmune sera. Noinhibition was detected with the preimmune sera.

Then, to assess whether P3 and 14F7 MAbs were ableto induce antibodies specific for gangliosides in thisanimal model, sera from immunized chickens weretested individually by ELISA for binding to purifiedNeuAc- and NeuGc-containing gangliosides. A strongand specific serological antibody response againstGM3(NeuGc) and GM2(NeuGc) gangliosides (titers1:8000) was detected by ELISA in the two chickensthat also developed antibodies against P3 MAb (datanot shown). HPTLC immunostaining confirmed the

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Fig. 4. Anti-idiotype and anti-isotype response induced in chickens immunized with P3 MAb (A) or 14F7 MAb (B). Hyperimmune

sera from a representative chicken immunized with P3 MAb (dilution 1/1000) (A), or 14F7 MAb (dilution 1/5000) (B), were

preincubated with the isotype-matched E1 MAb (A) or 1E10 MAb (B), and later the reactivity against P3 and E1 MAb (A), or 14F7

and 1E10 MAb (B), was assessed by ELISA.

A

B

0

10

20

30

40

50

60

50 100 200 400 800 1600 3200

1/dilution

% I

nhib

ition

HI vs 1E10

HI vs 3F9

HI vs 3B11

PI vs 1E10

PI vs 3F9

PI vs 3B11

010

2030

4050

6070

8090

100 200 400 800 1600 3200

1/dilution

% I

nhib

ition

HI vs 4G9

PI vs 4G9

Fig. 5. Inhibition of Ab1-Ab2 binding by sera from repre-

sentative chickens immunized with P3 MAb (A) or 14F7 MAb

(B), to their respective anti-idiotypic MAbs. Binding of anti-

idiotype antibodies (1E10, 3F9 and 3B11 MAb, A; and 4G9

MAb, B) to chimeric P3 (A) or chimeric 14F7 (B) coated

plates, was tested in the presence of serial dilutions of

preimmune (PI) and hyperimmune (HI) chickens sera.

Percentage of inhibition was calculated relative to binding of

P3Q (A) or 14F7Q (B) to the anti-idiotype MAb in the absence

of serum.


specificity of this anti-ganglioside antibody response(Fig. 6). Neither serum from chickens immunized with14F7 nor ior-c5 MAbs (isotype-matched control)recognized N-glycolyl gangliosides tested. Only P3-immunized animals showed a specific antibody responseagainst GM3(NeuGc) and GM2(NeuGc) gangliosides,in agreement with those obtained by ELISA.

Discussion

VH 14F7 is a member of the J558 (VHI) gene family.Kannagi et al. (1989) found that the majority of anti-ganglioside or anti-sulfated glycolipid antibodies use VH

genes from the J558 gene family, followed by J606 andQ52. Vk 14F7 is encoded by the gene 23–43 (Schaebleet al., 1999), which belongs to the Vk 23 gene family.14F7 is a highly mutated antibody with most of theamino acid replacements located in the VH region. Thisfinding agrees with reports by Chen et al. (1992) andDavid and Zouali (1995), who suggested that somaticmutations can take place separately in both variableregions (VH and VL) and, probably, at different times.In this aspect, 14F7 MAb differs from P3 MAb, which isa germline gene-encoded antibody (Perez et al., 2001).Both antibodies recognize GM3(NeuGc) ganglioside,but 14F7 MAb is the result of a mature humoralresponse against it, suggesting the involvement ofT-helper cells in the modulation of the response, whileP3 MAb is the result of a thymus-independent antigenresponse (Vazquez et al., 1995a). These differencesbetween P3 and 14F7 MAbs could relay on the way inwhich they were generated: P3 MAb was obtained byimmunizing BALB/c mice with GM3(NeuGc) ganglio-side embedded in liposomes together with tetanus toxoid

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Fig. 6. HPTLC immunostaining with sera from representative chickens: (A) Standard monosialogangliosides were chromato-

graphed with chloroform/methanol/0.2% CaCl2 in 2.5M ammonium solution and visualized with orcinol stain. The same

gangliosides as in A were chromatographed as above and immunostained with sera (dilution 1:500) from chickens immunized with

P3 (B), 14F7 (C) and ior-c5 (D) MAbs.


(Vazquez et al., 1995a), and 14F7 MAb was obtainedfrom mice immunized with GM3(NeuGc) gangliosidehydrophobically conjugated to human VLDL particlesand in the presence of Freund0s adjuvant (Carr et al.,2000). The increment of the immunogenicity of gang-liosides when they are conjugated to VLDL has beenpreviously described (Dummontet et al., 1997).

Besides, the H-CDR3 of P3 and 14F7 MAbs havedifferent immunogenetic origins. The D segment of VH14F7 is formed by the fusion of two D minigenesbelonging to the DSP2 family, and represents 33% ofthe H-CDR3, while this segment only comprises 17% ofVH P3 (Perez et al., 2001). Meek et al. (1989) havedemonstrated that the D–D fusion is not a property of aparticular set of D minigenes, and that these genes canbe used in both directions: 30–50or 50–30. Nevertheless,these antibodies have as a common feature in theirH-CDR3 the presence of arginine residues in the middlepart of the loop (Lopez-Requena et al., 2007a).

We have previously reported the importance of theelectronegative group present in the sialic acid residue ofthe gangliosides for the interaction with P3 MAb(Vazquez et al., 1995a; Moreno et al., 1998). Arginineresidues at positions 31, 98 and 100a in the VH segmentof P3 MAb, suggest this antibody could establishedhydrogen bonds and ionic interactions withGM3(NeuGc) ganglioside (Moreno et al., 1998). Fromthis point of view, the presence of basic residues inH-CDR3 of 14F7 MAb arranged in a motif Arg-X-X-Arg, similar to that found in P3 MAb (Perez et al.,2001), suggest to us the existence of a common patternof interaction with the electronegative group of the sialicacid. Modelling studies together with the crystalstructure of 14F7 MAb support the importance ofArg98 and Arg100a in the recognition of N-glycolyl sialicacid by this antibody (Krengel et al., 2004). Addition-ally, mutants of these residues on P3 MAb did not bindto the ganglioside (Lopez-Requena et al., 2007a).

Some of the initial studies in the field of antigen–anti-body interactions showed that cationic antigens developan anionic antibody response and, conversely, immuni-zation with anionic antigens generally induces a cationicantibody response (Sela and Mozes, 1966). 1E10 MAb isan anti-idiotypic antibody specific for P3 MAb (Vazquezet al., 1998). The immunogenetic analysis of 1E10 MAb

showed the presence of negatively charged residues in itsH-CDR3, located in the segment Asp96–Tyr97–Tyr98–Asp99 (Perez et al., 2001). The relevance of thecharged residues found in the H-CDRs of P3 and1E10 MAbs have been evaluated. When phage-dis-played random peptide libraries were faced to theseantibodies, the selected peptides were enriched inresidues of the opposite charge and in some cases withpatterns of spacing similar to those of the respectiveP3 and 1E10 MAb H-CDR3 (Perez et al., 2002;Lopez-Requena et al., 2007a). Additionally, H-CDR3from different Ab2 MAbs specific for P3 MAb displaysimilar complementary acidic acids (Perez et al., 2001;Lopez-Requena et al., 2007b).

4G9 MAb is an anti-idiotypic antibody generated inBALB/c mice immunized with 14F7 MAb coupled toKLH and emulsified in Freund0s adjuvant (Rodrıguezet al., 2003). The sequence of VH 4G9 revealed that thisMAb has a negatively charged segment in its H-CDR3(Tyr96–Asp97–Tyr98–Asp99). These acidic residues couldestablish electrostatic interactions with the basic resi-dues of 14F7 MAb H-CDR3, as also suggested for thebinding between P3 and 1E10 MAbs (Perez et al., 2001,2002).

Based on these findings, we assessed the possiblepresence of shared idiotopes in 14F7 and P3 MAbs.Neither sera from mice immunized with 14F7 MAbcross-reacted with P3 MAb, nor antibodies recognizing14F7 MAb were found in the sera from animalsimmunized with P3 MAb. In the same way, 1E10MAb do not react with 14F7 MAb and 4G9 MAb donot bind to P3 MAb (our unpublished data). Thus, theexistence of a similar basic residue motif in the HCDR3of P3 and 14F7 MAbs, was not enough to predict thepresence of common idiotopes. Further experiments arerequired to determine the importance of the motifArg98-X-X-Arg100a of the 14F7 MAb H-CDR3 for theinteraction with 4G9 MAb.

Perez et al. (2002) suggested that the Arg31 residue atthe H-CDR1 of P3 MAb could be part of a conforma-tional idiotope including also the motif Arg98-X-X-Arg100a of H-CDR3. The replacement of this residueby a serine residue showed its crucial role for theinteraction of P3 MAb with all its ligands (Lopez-Requena et al., 2007a). What is even more interesting,


it demonstrated the importance of this residue for P3idiotype antigenicity. Mutants carrying a serine at thatposition lost the immunodominance of the variableregion (Lopez-Requena et al., 2007a). Interestingly,14F7 MAb carries a serine residue at that position ofthe H-CDR1. However, this spatial arrangement ofbasic residues does not seem to be enough for explainingthe immunogenic differences between P3 and 14F7MAb, taking into account the low similarity of the restof the variable region amino acid sequences.

In addition, we demonstrated that 14F7 MAb isincapable of inducing an anti-idiotypic antibody re-sponse in the syngeneic model without coupling to acarrier protein. By contrast, P3 MAb induces a specificIgG anti-idiotypic antibody response when administeredin BALB/c mice, even in the absence of carrier proteinor adjuvant (Vazquez et al., 1998).

The generation of an Ab2 response in BALB/c miceimmunized with 14F7 MAb coupled to KLH, indicatedthe existence of anti-idiotypic B cells specific for thisantibody in the mouse repertoire, but this responsecannot be stimulated when 14F7 is administered onlyemulsified in Freund’s adjuvant. The immunogenicproperties have been correlated by several authors withsomatic mutations in the variable regions of theantibodies (Bogen et al., 1986; Hannestad et al., 1986;Wysocki et al., 1998). Nevertheless, we have demon-strated that 14F7 MAb, in spite of being a highlymutated antibody, does not fulfil this criterion. Then,neither the high immunogenicity of P3 MAb (Vazquezet al., 1998) nor the present results with 14F7 MAb canbe explained by the presence of somatic hypermutations.In fact, P3 MAb being of germline origin (Perez et al.,2001) is able to stimulate a T2 and a T3 cell response inBALB/c mice (Perez et al., 2002).

The immunogenicity differences of 14F7 and P3MAbs could also rely on their different isotypes. It hasbeen suggested that IgM antibodies are able to inducean Ab2 response in syngeneic animals more effectivelythan IgGs (Reitan and Hannestad, 1995). Severalexplanations were given for this phenomenon and oneof them is that mannose residues in the IgM structurecan stimulate their receptors in dendritic cells enhancingprocessing and presentation of IgM idiopeptides to Thelper cells. Moreover, it is well known that B cells bearFcg inhibitory receptors (FcgRIIb), which contain anITIM motif in their cytoplasmic tail that down regulatesAg-specific proliferation and differentiation of B cells(Cohen-Solal et al., 2004). Thus, the antibody eventuallyrecognized by a B cell receptor could also bind to Fcg-inhibitory receptors on the same B cell, abrogating theanti-idiotypic antibody response. This experimentalevidence could explain the absence of an Ab2 responsewhen 14F7 MAb is injected without a carrier protein.Nevertheless, in our experience, the IgM isotype is notenough to ensure the induction of anti-idiotypic

responses in the syngeneic model. In fact, other anti-ganglioside IgM antibodies generated by our group arenot immunogenic at all in BALB/c mice (Vazquez et al.,1998). Furthermore, the chimeric version of P3 MAbinduced in BALB/c mice an anti-idiotypic antibodyresponse stronger than the anti-isotypic one, a resultthat evidenced the immunodominance of the variableregion of P3 MAb (Lopez-Requena et al., 2003). Theimmunogenicity of P3 idiotype has also been demon-strated in the absence of any constant domain (ourunpublished results).

We have previously reported the induction of anN-glycolyl-ganglioside specific response in Leghornchickens immunized with Ab2 1E10 or 4G9 MAbs,thus behaving as ‘‘internal image’’ antibodies in thisanimal model (Rodrıguez et al., 2003; Hernandez et al.,2005), where N-glycolyl-containing gangliosides areforeign antigens. To define whether P3 and 14F7 MAbscould induce through an idiotypic cascade Ab3 with thesame specificity of the Ab1, they were also administeredto Leghorn chickens. Both antibodies were capable ofinducing a strong specific antibody response. Further-more, idiotypes from P3 and 14F7 MAbs wereimmunogenic in this xenogenic model, considering theanti-idiotype response after the absorbing of isotyperesponses with an irrelevant antibody. The hyperim-mune sera were effective in inhibiting the Ab1 binding totheir respective Ab2s, thus evidencing the presence ofshared idiotopes. But they differ in their capacity tostimulate an anti-anti-idiotypic (Ab3) response againstNeu-containing gangliosides. In contrast with the resultsobtained for 14F7 MAb, P3 MAb was capable ofinducing specific Ab3 antibody response againstGM3(NeuGc) and GM2(NeuGc) gangliosides. Thisdifference could be explained due to the quality of thegenerated response. It means 14F7 Ab1 MAb couldpredominantly induce an Ab2g response, which interactswith an idiotype shared by 4G9 Ab2 MAb that can notmimic the antigen. A second explanation could be thehigh connectivity of the immune network generated bygermline encoded antibodies (Varela and Coutinho,1991), such as the immunogenetic analysis of P3 MAbhas suggested (Perez et al., 2001). Our experimentalresults fit in general terms with these hypotheses.However, further experiments to understand the mole-cular basis of the idiotypic immunogenicity of P3 MAbare now ongoing.

Acknowledgments

We thank Dr. L.E. Fernandez (Vaccine Department,Center of Molecular Immunology) for generouslyproviding gangliosides. This study was supported bythe Cuban Government.


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