Gangliosides, Ab1 and Ab2 antibodies III. The idiotype of anti-ganglioside mAb P3 is immunogenic in a T cell-dependent manner.

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 gamma1CH3, 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.

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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-Requenaa,b, Marco Bestagnob, Cristina Mateo de Acostaa,
Michela Cesco-Gaspereb, Ana Mar´ ıa V´ azqueza, Rolando P´ ereza, Oscar R. Burroneb,∗
aDepartment of Antibody Engineering, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba
bMolecular 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
showntobeappropriatetoinducespecificanti-idiotypicresponses.Bygenegunimmunization,apolyspecificresponsewasoccasionallygenerated,
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.
TherequirementofTcellsintheanti-P3idiotyperesponsewasindicatedbythelackofP3-specificanti-idiotypicantibodiesfollowingimmunization
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
Theimmunogenicityofautologousimmunoglobulinsisnota
frequentlydescribedphenomenon.BoththeIgMisotype(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: burrone@icgeb.org (O.R. Burrone).
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 anti-
body 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).
P3isabletoinduceaverystronganti-idiotypicAb2response
in syngeneic BALB/c mice, even when administered without
carrier proteins or adjuvants (V´ azquez et al., 1998). Immuno-
genetic analysis of P3 and 1E10 mAb variable regions (P´ erez
et al., 2001) as well as the selection of peptide mimotopes from
phagelibraries(P´ erezetal.,2002;Lopez-Requenaetal.,2007a),
ledtotheidentificationofsomekeybasicresiduesonP3H-chain
CDRs 1 and 3 that seemed to be complementary with a motif
0161-5890/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.molimm.2007.01.010

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A. L´ opez-Requena et al. / Molecular Immunology 44 (2007) 2915–2922
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 immuno-
genicity. In fact, other anti-ganglioside IgM tested were unable
toinduceanAb2responseintheabsenceofadjuvants(V´ azquez
etal.,1998).Inthepresentwork,weuseDNAimmunizationand
small immune proteins (SIPs) (Li et al., 1997) for assessing the
contribution of constant regions for the immunogenicity of the
P3idiotype.SIPsarebivalentproteinsbasedonscFvfragments,
where the two variable regions of the H and L chains, VH and
VL,respectively,areconnectedwithatetrapeptidetothemurine
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
DNAimmunizationeitherbygenegunorbyrecombinantadeno-
associated virus (rAAV) infection. The requirement of T cell
activation for the anti-idiotypic response was demonstrated.
2. Materials and methods
2.1. Cells
Sp2/0 and HEK293 cells from American Type Culture Col-
lection (ATCC) (Rockville, MD) were cultured in RPMI 1640
or Dulbecco’s modified Eagles medium (DMEM) (Gibco, Pais-
ley, UK), respectively, supplemented with 10% heat inactivated
fetal calf serum (FCS) (Gibco), gentamycin (50?g/mL), and
2mM 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.
2.2. Animals
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 barrier-
maintained room according to standard animal care and use
guidelines.
2.3. Ganglioside and chimeric antibodies
GM3(NeuGc) ganglioside was kindly provided by Dr. L.E.
Fern´ andez,VaccineDepartment,CenterofMolecularImmunol-
ogy (Havana, Cuba).
Chimeric P3, 1E10 (Lopez-Requena et al., 2003) and P3 H-
CDR mutant (Lopez-Requena et al., 2007a) antibodies (human
IgG1, ?) were purified from transfectoma culture supernatants
by Protein-A Affinity Chromatography (Pharmacia, Uppsala,
Sweden) and analyzed by SDS-PAGE under reducing condi-
tions. The specificity of the purified antibodies was confirmed
byenzyme-linkedimmunoadsorbentassay(ELISA)andprotein
concentration estimated by optical density at 280nm.
2.4. Primers
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?-ACAGGTGCACTCGGACATTGTGATGACC-
3?; 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?.
ForthetransferofthewholeSIPcassettetothepAAVvector,
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?.
2.5. Vectors
The pUT-SEC vector for the construction of the scFv frag-
ment has been previously described (Li et al., 1997). The
pcDNA3-?1CH3(murine)(Lietal.,1997)andpcDNA3-?CH4-
sEMPD-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 prox-
imal 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).
2.6. SIP construction and expression
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 pUT-
SEC vector, digested XhoI/BspEI for the VH or ApaLI/SpeI for
theV?.AfragmentHindIII/BspEIcomprisingthescFvfragment
withaleaderpeptidewasthentransferredintotheSIPpcDNA3-
based vectors equally digested. Sp2/0 cells were transfected by
electroporation as previously described (Bestagno et al., 2001).
TheselectionofstableSIP-secretingcloneswasdonebyELISA.
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

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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 450nm. Stable memb-SIP-displaying trans-
fectomas 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 incu-
bation 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
afterthemurine?1CH3domainintotheXbaI-digestedpcDNA3
vector, and the whole SIP fragment was then cloned into the
HindIII/BglII-digestedpAAV-MCSvector.rAAVparticlespseu-
dotyped with the capsid serotype 8 were obtained at the AAV
vector unit of the International Centre for Genetic Engineer-
ing and Biotechnology (ICGEB), Trieste, Italy. Virus titration
was performed by real-time PCR. Preparations had approxi-
mately (2–4)×1011units/mL. For checking SIP expression and
functionalitybyELISA,HEK293cellswereinfectedwithdiffer-
ent multiplicities of soluble SIP-coding rAAV and supernatants
taken after 72h.
2.8. Mice immunization
The antibody response induced in mice by the SIPs was
determined by DNA immunization, either by gene gun or rAAV
infection.Forgenegunexperiments,BALB/c,athymicBALB/c
andC57BL/6micewereimmunizedintradermallybythreeshots
at 2-week intervals with the SIP constructs. Mice abdominal
areaswereshavedand1?mgoldparticlescarrying1–3?gDNA
were injected at 400psi using the BioRad gene delivery device
(Biorad, Hercules, CA). Sera were collected 10 days after the
lastshot.AdditionalBALB/cgroupsreceivedonlyoneshot,and
sera were extracted 10, 38 and 101 days after. rAAV infection
of BALB/c mice was performed by inoculating intramuscularly
in both legs 3×1010viral particles. Serum collection was done
30 days after the infection.
2.9. Antibody binding assays
Binding of SIPs to GM3(NeuGc) ganglioside was deter-
mined using an ELISA assay as previously described (Alfonso
et al., 1995; Lopez-Requena et al., 2007a), with the super-
natants 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 deter-
mined 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.
Tomeasureanti-SIPreactivityinserafromimmunizedmice,
the microtiter plates were coated with 10?g/mL of the chimeric
antibodies or human IgG purified from normal serum (Sigma).
AfterblockingtheplateswithPBS-T-BSA,1:100dilutedserum
samples were added and incubated for 2h at 37◦C, and alkaline
phosphatase-conjugated anti-mouse IgG (Pierce) was used as
secondantibody.Somechosenserawerealsotestedforreactivity
with memb-SIPs, by FACS. Memb-SIP-displaying transfec-
tomas 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 coef-
ficient of variation was <10% for all assays. Background values
of absorbance were less than 0.1.
3. Results
The idiotypes of P3 and P3 mutants assembled as scFvs in
the murine SIP format were expressed and tested for their bind-
ing 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 anti-
body (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
recognizedbycytofluorimetrybyantibodies1E10orP3,respec-
tively (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 differ-
ent 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 anti-
bodies that were not specific, but showed reactivity against all
antibodies, including polyclonal human IgG (Fig. 2A). In con-
trast, animals immunized with the 1E10-SIP, induced a highly
specificanti-idiotypicresponse(Fig.2B).However,whentested
by FACS on cells expressing the P3-memb-SIP or the 1E10-
memb-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, respec-
tively (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-idiotypicantibodiesagainsttherespectiveimmunizingidio-
type, 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 corre-
spondingtotheP3-SIPandthosefromthefourmutants(datanot
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.
animalsimmunizedwiththepurifiedprotein(Lopez-Requenaet
al.,2007a),serafromanimalsimmunizedwiththeSIPconstruct
DNA of the R31→S mutant produced antibodies recognizing
theP3idiotype.WeconfirmedtheseresultswithserafromDNA
or protein-immunized animals by cytofluorymetry on cells dis-
playing 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
ofplasmidDNA(Fig.6).Inthiscondition,onlyP3-SIPinduced
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 P3-
or 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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2919
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. Further-
more, 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).
Inordertofurtherinvestigatethenatureoftheimmunogenic-
ity of the P3 idiotype, we tested the anti-idiotypic response in
animals from the allogeneic C57BL/6 strain (H2bhaplotype)
and in athymic BALB/c mice. As shown in Fig. 7, the reactiv-
ity against chimeric P3 (Fig. 7A) or 1E10 (Fig. 7B) antibodies
was practically absent in the sera from C57BL/6 animals immu-
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
purifiedchimericR31→Santibody.Serawereassayedat1:100dilution.Arrows
indicatetheimmunogenineachcase.Filledcurve,preimmuneserum.Reactivity
wasdeterminedusingaFITC-conjugatedgoatanti-mouseIgG(?-chainspecific)
antibody.
nizedwiththerespectiveSIPs.Moreimportantly,asimilarresult
was obtained with the athymic BALB/c mice (Fig. 7C and D).
Takentogethertheseresultssuggestthat:(i)acompleteimmuno-
competent 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 com-
patible 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 recom-
binant adeno-associated virus (rAAV) delivery of the P3-SIP
construct,asanalternativewayofDNAimmunization.Infective
rAAV are produced in mammalian cells and therefore, contrary
to the bacterial plasmid DNA, the viral DNA does not contain
hypomethylated CpGs.
AsshowninTable1,theP3-SIPconstructdeliveredbyrAAV
infectioninducedananti-idiotypicspecificantibodyresponsein
Fig.6. IgGresponseinBALB/cmiceimmunizedoncebygenegunwiththeP3-
or 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
P3-SIP 1E10-SIP
ch P3 Ab
ch 1E10 Ab
3/9
0/9
0/9
0/9
aResults represent number of mice with reactivity (at least twice the preim-
mune 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 con-
struct 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 anti-
body (Carr et al., 2000), requires coupling to keyhole lympet
haemocyanin(KLH)andemulsificationinFreund’sadjuvantfor
eliciting an anti-idiotypic response in BALB/c mice (Rodr´ ıguez
etal.,2007).P3mAbisagerm-lineIgM(P´ erezetal.,2001)while
14F7 is a hypermutated IgG (Rodr´ ıguez et al., 2007). Immuno-
genicity of P3 was also verified with the chimeric version of
the molecule, because anti-idiotypic antibodies were the main
componentofthehumoralresponseinmiceimmunizedwiththe
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
previousexperimentshadbeenadministeredaspurifiedprotein.
TheabsenceofresponseagainstthepeptidelinkerofthescFvin
theSIPconstructshaspreviouslybeenreported(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
coupledtoKLHandemulsifiedinFreund’sadjuvant(V´ azquezet
al.,1998).Nevertheless,theanti-idiotypicresponsewasimmun-
odominant not only in the case of mice immunized with the
chimericantibody(Lopez-Requenaetal.,2003),butalsoforthe
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 immuniza-
tion of antibodies able to recognize proteins with the human
IgG isotype. This response seemed to depend on hyperimmu-
nization, because it was not observed in mice that received
only one shot of the P3-SIP-coding plasmid. Antibodies gen-
erated 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 pres-
ence of such antibodies could not be demonstrated. As it was
alsoobservedthatthemutantantibodylosttheidiotypicimmun-
odominance or antigenicity of the wild type version, it is still
possible that P3 idiotype-specific antibodies could have been

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2921
generated but not detected at the dilution assayed. This expla-
nation is consistent with the fact that antibodies specific for the
original idiotype could be detected only in the sera with the
highesttitersfromanimalsimmunizedwiththeH-CDR3R→S
mutants (Lopez-Requena et al., 2007a). Also, the differences in
antibody titers observed in animals immunized with the puri-
fied mutant chimeric antibodies were practically absent in mice
immunized with the corresponding SIP DNA.
Bacterial DNA immunization could be related to these unex-
pected antibody responses. Hypomethylated CpG sequences,
typicalofbacterialDNA,stimulatecellsexpressingtheToll-like
receptor(TLR)9(Hemmietal.,2000;Wagner,2004).Thisinter-
action initiates an immunomodulatory cascade including the
activation of B and T lymphocytes, “natural killer” cells, mono-
cytes, macrophages and dendritic cells (Ashkar and Rosenthal,
2002; Klinman, 2004). However, clear differences in the speci-
ficity of the anti-idiotypic antibodies induced with protein as
opposedtoDNAimmunizationshavebeenpreviouslydescribed
(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 serocon-
version was demonstrated. While plasmid vectors are amplified
inbacteria,therecombinantviralparticlesareproducedinmam-
malian 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
reproducedeitherintheallogeneicC57BL/6modelorinathymic
BALB/c mice. These results strongly suggest the involvement
ofhelperTcellsinthisresponse,inviewoftheMHC-H2haplo-
type differences between both strains and the absence of T cells
intheathymicanimals.Thisisconsistentwithpreviousobserva-
tions 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 asso-
ciate with MHC class II molecules of the H2dhaplotype and
initiate a T helper response to assist the anti-idiotypic specific
B cells. Although the gene gun immunization could have con-
tributed to an enhanced efficiency of the anti-idiotypic response
by activating TLR in antigen-presenting cells, as observed for
theanti-1E10idiotyperesponse,theintrinsicimmunogenicityof
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 idio-
types with the unusual properties of P3 in the repertoire of
neonatal mice as well as in healthy or autoimmune adults.
Acknowledgements
We thank Dr. M. Poggianella from ICGEB for valuable
advices and Mauro Sturnega (ICGEB) and Tania Gri˜ n´ an (CIM)
for excellent technical assistance. We are also grateful to Dr.
M. Giacca, Dr. L. Zentilin, Marina Dapas and Michela Zotti for
rAAV preparation. This work was supported by ICGEB and the
Cuban government.
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