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Molecular Immunology 47 (2009) 270–282

Contents lists available at ScienceDirect

Molecular Immunology

journa l homepage: www.e lsev ier .com/ locate /mol imm

ariable epitope library-based vaccines: Shooting moving targets

esar Pedroza-Roldana, Claudia Charles-Ninoa, Rafael Saavedraa, Tzipe Govezenskya,uis Vacab, Eric Avaniss-Aghajanic, Goar Gevorkiana, Karen Manoutchariana,∗

Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), AP 70228, Cuidad Universitaria, México DF 04510, MexicoInstituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM), AP 70228, Cuidad Universitaria, México DF 04510, MexicoPrimex Clinical Laboratories, Inc, 16742 Stagg St. Unit 120, Van Nuys, CA 91406, USA

r t i c l e i n f o

rticle history:eceived 16 July 2009ccepted 9 September 2009vailable online 23 October 2009

eywords:pitope vaccinepitope librariesmmunogenntigenic variabilityIV vaccine

mmune interferenceouse

a b s t r a c t

While the antigenic variability is the major obstacle for developing vaccines against antigenically vari-able pathogens (AVPs) and cancer, this issue is not addressed adequately in current vaccine efforts. Wedeveloped a novel variable epitope library (VEL)-based vaccine strategy using immunogens carrying amixture of thousands of variants of a single epitope. In this proof-of-concept study, we used an immun-odominant HIV-1-derived CD8+ cytotoxic T-lymphocyte (CTL) epitope as a model antigen to constructimmunogens in the form of plasmid DNA and recombinant M13 bacteriophages. We generated combi-natorial libraries expressing epitope variants with random amino acid substitutions at 2–5 amino acidpositions within the epitope. Mice immunized with these immunogens developed epitope-specific CD8+IFN-�+ T-cell responses that recognized more than 50% of heavily mutated variants of wild-type epi-tope, as demonstrated in T-cell proliferation assays and FACS analysis. Strikingly, these potent and broadepitope-specific immune responses were long lasting: after 12 months of priming, epitope variants wererecognized by CD8+ cells and effector memory T cells were induced. In addition, we showed, for the first

time, the inhibition of T-cell responses at the molecular level by immune interference: the mice primedwith wild-type epitope and 8 or 12 months later immunized with VELs, were not able to recognize variantepitopes efficiently. These data may give a mechanistic explanation for the failure of recent HIV vaccinetrials as well as highlight specific hurdles in current molecular vaccine efforts targeting other importantantigenically variable pathogens and diseases. These findings suggest that the VEL-based strategy forimmunogen construction can be used as a reliable technological platform for the generation of vaccines

and c

against AVPs and cancer,

. Introduction

The extensive genetic and antigenic variability is the majorbstacle for the development of urgently needed vaccines againstIV/AIDS, HCV, influenza, malaria and cancer Tobin (Tobin et al.,008). Despite this, the issue of antigenic diversity has not beenddressed adequately to this date, or even directly, in modernaccine concepts and, the proposed strategies for immunogen con-truction lack convincing elements of rational design specificallyargeting genetic/antigenic variability. Perhaps, the most dramaticase of vaccine failure represents HIV/AIDS, considering the enor-ous investments in financial and human resources (Walker and

urton, 2008; Barouch, 2008).There is compelling evidence that CD8+ T cells are key com-

onents of immune responses against intracellular pathogens andumors and, those effective vaccines against AVPs will likely need to

∗ Corresponding author. Tel.: +52 5556229194; fax: +52 5556229212.E-mail address: karman@servidor.unam.mx (K. Manoutcharian).

161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.molimm.2009.09.024

ontribute to better understanding complex host–pathogen interactions.© 2009 Elsevier Ltd. All rights reserved.

induce broad and potent cellular responses (Appay et al., 2008). Theepitope recognition in MHC-restricted T-cell responses involvestwo different binding events each acting as possible limiting fac-tors: first, the epitope peptides bind to the MHC molecules; second,the resulting peptide-MHC (pMHC) complex is bound by T-cellreceptor (TCR) leading to cell activation (Sparbier and Walden,1999). The important feature of any epitope is its dominance aswell as degenerate and promiscuous nature of TCR–pMHC inter-actions resulting in TCR cross-reactivity: the ability of a TCR torecognize several different peptides bound to self-MHC molecules(Nikolich-Zugich et al., 2004).

Just the presence of activated T cells is not sufficient to containHIV, for example and, the recent advances in the characterization ofT-lymphocyte responses by multiparameter flow cytometry havehighlighted the functional diversity of virus-specific T cells in terms

of cytokine secretion, degranulation, proliferation, antigen sensi-tivity and other effector functions in various subpopulations ofeffector and memory T cells (Barouch, 2008; Almeida et al., 2009).Unfortunately, there are several mechanisms allowing the virusto escape the immune attack by T cells, such as antigen sequence

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ariation, altered antigen presentation, latency, privileged sites ofiral replication, as well as mechanisms causing the loss of effectorells or affecting their functional integrity (McMichael and Hanke,002). However, the extreme antigenic variability of HIV causedy high mutation and recombination rates of the virus leading to

mmune escape is the major obstacle for the successful design ofvaccine (Altfeld and Allen, 2006; Yang et al., 2005; Brander et

l., 2006). Mutations of amino acids within epitopes or in epitopeanking regions abrogate MHC binding or interfere with effectiventigen processing, respectively, preventing subsequent presenta-ion of the epitope to T cells (Tenzer et al., 2009).

The CTL escape epitope might also revert back to wild-type,hich occurs very fast in early infection, if significant fitness costs

re incurred by the mutation (Matthews et al., 2008; Wang etl., 2009). Transmission and accumulation of CTL escape variantseflects the balance between CTL immune pressure, antigenic vari-bility of HIV and HLA polymorphism of the host, linking thearticular HLA allele, including those strongly associated with theontrol of infection, with preservation of consensus viral sequencesLeslie et al., 2005; Crawford et al., 2009). However, the rapid pro-ess of viral adaptation may dismantle known HLA associationsith control of HIV infection that are linked to the availability of

ey epitopes, and highlights the challenge for a vaccine to keep paceith the changing immunological landscape presented by the virus

Kawashima et al., 2009). Importantly, if an escape variant reachesxation in the population, the epitope will be lost as a potentialaccine target. So, it is suggested that inducing synchronous, broadTLs by vaccination should limit the likelihood of viral escape from

mmune control (Brander et al., 2006; Davenport et al., 2008).The current HIV vaccine efforts are based mainly on immuno-

ens bearing conserved, consensus, ancestral or centre of treeegions of HIV (Weaver et al., 2006; Santra et al., 2008), mosaicFischer et al., 2007) or scrambled protein sequences (Thomson et

l., 2005), combinations of antigens derived from major HIV cladesLetourneau et al., 2007), with the hope to induce the broadest pos-ible activation of immune cells capable of recognizing diverse viraltrains and epitopes. However, most of such consensus sequences

ig. 1. Generation of immunogens based on variable epitope libraries (VELs). Schematonstruct and as recombinant M13 bacteriophages (not in scale). (A) Eight partially overlaPCR assembly to generate a DNA fragment. (B) The DNA was amplified by PCR using primII sites of VHExpress vector resulting in VVH control plasmid. The oligo B7 encoding HCespectively, in two separate PCR assembly/amplification and generated DNAs were similNA constructs. (B and C) The DNA fragments encoding LWT and VEL without Ig-VH contnd cloned into phagemid vector pG8SAET generating LWT- and VEL-expressing phage c

munology 47 (2009) 270–282 271

of HIV and other AVPs have been already naturally selected by thehost immune system as antigenic regions lacking protective epi-topes, thus, even when epitopes derived from conserved sequencesare immunogenic and may be recognized by the host, they can-not induce protective immunity by definition. Otherwise, the AVPscarrying “true” protective epitopes simply should disappear fromcirculation as a result of competitive exclusion or, such epitopesmay reside only in most antigenically variable regions of AVPs.Indeed, there is clear experimental evidence showing that themajority of currently circulating viruses are not recognized byhuman T cells (Altfeld et al., 2005) as a result of a mutation withinotherwise immunodominant CTL epitope. Also, immunocompe-tent macaques challenged with a simian immunodeficiency virus(SIV) bearing common escape mutations in CTL epitopes recognizedpoorly these epitope variants (Friedrich et al., 2004).

Several alternative HIV vaccine design approaches were pro-posed pretending to achieve high degree of immunologicalcross-reactivity between vaccine immunogens and viral epitopes.Thus, vaccine concepts were developed, termed as hypervariableepitope constructs (HECs) (Anderson et al., 2005), multiple epitopeimmunogens (MEIs) (Hewer and Meyer, 2003) or mixotope peptidelibraries (Oliveira et al., 2005) all based on the generation of epi-tope/peptide libraries or peptide cocktails where most frequentlyfound natural amino acids were included in defined amino acidpositions of peptides. However, such immunogens have the samedrawbacks as above-mentioned HIV vaccines based on conservedviral sequences and, again, their success is unlikely. Indeed, the HECvaccine had no antiviral effect in a pilot study where vaccinatedmacaques were challenged with SIV (Anderson et al., 2005).

In order to explore new and more rational approaches to con-tend with AVP or diseases (such as cancer), we developed a novelimmunogen construction and vaccine development concept basedon a simple and fundamental requirement for any vaccine: to be

successful, a vaccine should match antigenically the pathogen andthe disease condition at epitope level and should be capable ofinducing immune responses that resemble responses induced bythe pathogen itself. In this proof-of-concept study, we present data

ic representation of the design and generation of VELs as plasmid-encoded DNApping oligonucleotides (B1–B8), collectively encoding Ig-VH domain, were used iners 5Namp and 3Namp, digested with Pst I/Bst EII and, (C) introduced into Pst I/BstDR3 was replaced by oligo LN or oligos L1 + L2 (Table S1), encoding LWT and VEL,

arly cloned into VHExpress eukaryotic expression vector leading to VHPL and VHBLext were amplified using primers 5Damp and 3Damp, digested with Nco I/Bam HI

lone ELN and phage-displayed BFL library, respectively.

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n immunogenic properties of novel vaccine components basedn HIV-1 immunodominant CTL epitope-related VELs in mice. Theesults obtained are encouraging, strongly suggesting that thisovel approach may serve as guideline to develop new vaccineroduction strategies.

. Materials and methods

.1. Immunogen construction

All molecular biology procedures were carried out using stan-ard protocols or as recommended by manufacturers. Restrictionnzymes, DNA isolation/purification kits, T4 DNA ligase and13KO7 helper phage were obtained from, Promega (Madison, WI,SA) Invitrogen (Carlsbad, CA, USA), Qiagen (Valencia, CA, USA) oribcoBRL (Rockville, MD, USA).

The general scheme of immunogen construction is shown inig. 1. To generate DNA constructs expressing HIV-1 (clade B, IIIBsolate) gp120 V3-derived CTL epitope (epitope L) (aa 311–320;GPGRAFVTI) and VELs, we inserted the epitopes into human

mmunoglobulin (Ig) heavy-chain variable (VH) domain by replac-ng complementarity-determining-region 3 (HCDR3) of VH by CTLpitope and VELs, as we have reported previously (Manoutchariant al., 1999). First, to generate a wild-type (WT) Ig-VH domain, a setf partially overlapping oligonucleotides, collectively coding for theramework (FR) and CDR regions of the human Ig-VH domain DP47Fig. 1, oligos B1–B8, and Table S1), were synthesized at Operonechnologies, Inc., Alameda, CA. Oligonucleotides B1 to B8 (20 pmolach; the overlaps between the complementary oligonucleotidesre 12–20 nucleotides) were combined and assembled in a PCRith platinum Taq DNA polymerase (Invitrogen, USA) by cycling

he reaction mixture (25 �l) (95 ◦C for 7 min); (7 times 95 ◦C for.5 min; 56 ◦C for 2 min; 72 ◦C for 2 min). An aliquot from this reac-ion (0.5 �l), containing a 350-bp DNA fragment coding for the WTg-VH domain was amplified by PCR (50 �l) by cycling 30 times94 ◦C for 1 min; 60 ◦C for 1 min; 72 ◦C for 1 min) with the 5NAmpnd 3NAmp primers (25 pmol each), which introduce Pst I and BstII restriction sites at the 5′ and 3′ ends of the synthesized Ig-VHomain, respectively (the restriction sites are underlined in oligos,able S1). The assembly and amplification of PCR products werehecked by agarose gel electrophoresis, and the DNA of the engi-eered VH domains, after purification from the gel with a WizardV Gel and PCR Clean-up Systema (Promega), was digested with Pstand Bst EII restriction enzymes and purified again. Then, 100 ngf this DNA was ligated with 10 U of T4 DNA ligase (Amersham-ife Science, Cleveland, OH) to approximately 50 ng of Pst I- and BstII-digested DNA of the VHExpress eukaryotic expression vectorPersic et al., 1997) to generate control plasmid DNA designateds VVH. The ligated DNA was used to transform Escherichia coliG-1 cells by electroporation using Gene Pulser II System (Bio-ad Laboratories, Inc., Hercules, CA, USA). We verified the correctCR assembly and cloning by DNA sequencing performed on aenetic Analyzer ABI PRISM 3100 (Applied Biosystems, Foster City,A, USA) using miniprep-purified (Qiagen Inc., Santa Clarita, CA,SA) double-stranded DNA and the primers 5NAmp and 3NAmp

Table S1).Similarly, to generate modified VH domains expressing epi-

ope L and L epitope variants bearing VEL, referred as VHPL andHBL, respectively, the same mixture of oligos B1–B8 were used

n two separate PCRs by replacing B7 oligo, coding for HCDR3egion, with oligos LN and equimolar mixture of oligos L1 and L2

degenerate oligos where K in NNK triplets are T or C nucleotide),ncoding epitope L and epitope library, respectively (Fig. 1 andable S1), and using the same 5NAmp and 3NAmp primers asescribed above. The DNA fragments were inserted into VHEx-ress vector and, to generate VEL-expressing DNA construct VHBL,

munology 47 (2009) 270–282

10 electroporations were performed, and the transformed TG-1cells were plated on Luria Broth-Amp (LB) (50 �g/ml) plates todetermine the diversity of the library. The plasmid DNA was pro-duced by growth in E. coli (strain TG-1) in yeast extract tryptonemedium (YT2X) with Amp (50 �g/ml) and purified using QiagenMegaPrep columns, according to the manufacturer’s directions(Qiagen, Valencia, CA).

In order to express the L and L epitope variants bearing VELs onM13 phage surface as fusions with major phage coat protein (cpVIII)at high copy numbers, the corresponding DNA fragments havebeen cloned in pG8SAET phagemid vector as previously described(Manoutcharian et al., 2004). The epitopes are flanked by 5 aminoacids from FR3 and FR4 of VH domain. First, the DNA fragmentshave been generated by two separate PCRs using primers 5DAmpand 3DAmp carrying Nco I and Bam HI restriction sites (underlinedin oligos, Table S1 and Fig. 1) and PCR-assembled DNA encod-ing WT L epitope and L-based VEL, respectively, as templates.Then, these DNAs were purified and used in separate ligation reac-tions with similarly digested DNA of pG8SAET phagemid vectoras described above. After electroporation, the transformed TG-1cells were plated on LB-Amp plated to determine the diversityof the library. The resultant recombinant phagemid clone ELNexpressing L epitope and phagemid library carrying L-based VEL,referred as BFL, were rescued/amplified using M13KO7 helperphage, purified by double PEG/NaCl (20%, w/v polyethylene glycol-8000; 2.5 M NaCl) precipitation and resuspended in Tris-bufferedsaline (TBS). By the same way, three additional phage librariesBFL4, BFL3 and BFL2 carrying VELs with 4, 3 or 2 randomizedamino acid positions, respectively, were constructed using oli-gos L4, L3 and L22, respectively (Table S1). Also, a phage clone,referred as B1, expressing non-related epitope/peptide ASQFLLETQwas generated, corresponding synthetic peptide NRP was preparedand used as control antigens. As a control phage immunogen,a non-related phage (GK1) similarly expressing Taenia crassicepsrecombinant antigen-derived peptide GK1 (Manoutcharian et al.,2004) (aa 69–95: GYYYPSDPNTFYAPPYSA) at high copy numberson phage surface was generated.

The typical phage yields were 1010 to 1011 colony-forming units(cfu) per milliliter of culture medium. The generated recombinantphage particles were used as immunogens/antigens in immuniza-tion and lymphoproliferation assays. 20 phage-displayed epitopevariants have been randomly selected from BFL epitope library andused as antigens in T-cell activation assays. The DNA from thesephage clones were sequenced, the amino acid sequences of peptideinserts were deduced (Table 1) and corresponding peptide insertswere prepared as synthetic peptides and, similarly, used as antigensin T-cell assays. The synthetic peptides corresponding to epitope Ldesignated as PNL, non-related epitope B1 (PB1) and 10 epitopevariants, randomly selected from BFL phage library, were preparedat GenScript Corporation (NJ, USA).

2.2. Mice and immunizations

Groups of 3–5 female, 4–6-week-old BALB/c mice were used.Mice were immunized with L epitope or VEL-bearing constructsin the form of plasmid DNA or M13 phage particles. 1 × 1011

recombinant M13 phage particles (1 × 1011 colony-forming units,CFU) were used to immunize mice by intraperitoneal (i.p.) injec-tion. When the DNA vaccine was used, groups of mice wereimmunized by intradermal (i.d.) injection with 75 �g of plasmidDNA in 100 �l of saline buffer. Also, the groups of mice were

immunized with control vector VVH DNA construct and with con-trol GK1 phage. Separate groups of mice were primed by i.d.DNA injection with VHBL, VHPL or VVH and boosted 8 or 12months later with VHBL, and used in immune interference stud-ies.

C. Pedroza-Roldan et al. / Molecular Immunology 47 (2009) 270–282 273

Table 1Sequences of LWT epitope variants.

L epitope R Ga Pa G R A F V T I

Sequence design Xb G X G X A X V X IEpitope variantsL1 F – D – I – G – F –L2c D – I – S – G – G –L3 F – D – T – A – F –L4 T – T – T – C – S –L5 I – G – V – M – M –L6 S – G – G – G – G –L7 D – R – C – A – R –L8 S – E – S – G – L –L9 – – G – G – P – G –L10 T – I – A – P – C –L11 Y – N – W – A – V –L12 A – K – G – L – Q –L13 G – F – G – V – I –L14 F – D – T – G – F –L15 L – – – S – W – L –L16 – – C – S – G – R –L18 E – I – C – G – R –L19 – – L – V – V – V –

Sequence design R G P G X A X X X XL17 – – – – – – R L S VL20 – – – – G – S – L E

Amino acid frequencies 11/18 12/18 9/20 10/20 11/20

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a N-terminal MHC anchor positions, the amino acid Glycine (G) at anchor position3 was mutated.b X – any of 20 natural amino acids.c Epitopes marked in bold were prepared also as synthetic peptides.

.3. Proliferation assays

Spleen cells were obtained from individual mice on day5 after immunization and tested in a standard [3H]thymidine

ncorporation assay as total splenocytes. Cells were resus-ended in RPMI medium supplemented with 10% FBS, 1%enicillin/streptomycin, 1% sodium pyruvate, 1% nonessentialmino acids, 1% 2-mercaptoethanol. Effector cells were culturedn a 96-well flat-bottom plate (5 × 104 cells/well) with 1 × 1010

hage particles or 10 �M synthetic peptide corresponding to par-icular epitope variant. Culture was incubated for 72 h (3 days) inhe case of phage stimulation and 120 h (5 days) in the case ofeptide stimulation, the cells were pulsed for 16 h with 0.5 �Ci of

3H]thymidine per well. The cells were harvested on glass filteraper and incorporated radioactivity was measured with a liquidcintillation counter. The mean number of counts per minute (cpm)rom triplicate wells was used to calculate the stimulation index (SI)s follows: SI = cpm with antigen/cpm with medium alone. Simi-arly, spleen cells were obtained from mice primed and boosted byNA immunization and used in T-cell proliferation assays.

.4. Flow cytometric analysis

CD4+ and CD8+ T-cell responses were evaluated by using intra-ellular staining (ICS) for IFN-�; pooled splenocytes (5 × 106 cells)ere stimulated by 3 (phage antigen) or 5 days (peptide antigen)

s described in proliferation assay. Monensine was added to theells and incubated for 6 h. Cells were fixed and permeabilized with

Inside Stain Kit” (Miltenyi Biotec) according to manufacturer’snstructions. The cells were washed with phosphate-buffered salinePBS) and staining with indicated fluorescence-labeled monoclonalntibodies against CD4, CD8 (BD, Pharmingen, USA) for 30 min

n ice. After washing, the cells were permeabilized with 0.1%aponin and labeled for 10 min with anti-IFN-� in the dark. Mem-ry response was evaluated using anti-CD3, CD8, CD44 and CD62L.he cells were analyzed by FACS Calibur Cytometer and CellQuestoftware; at least 10,000 events were collected. Pooled cells from

s left intact in both versions of mutated positions, the Proline (P) at anchor position

groups of mice (n = 3) were used and two independent experimentswere carried out.

2.5. Statistical analysis

Data were expressed as means ± standard deviations of themeans (SD). Mann–Whitney two tailed t test and Kruskal–Wallistest were performed using GraphPad Prism version 4.0 for win-dows. P-value less than 0.05 was considered significant.

3. Results

3.1. Immunogen construction

We generated recombinant M13 phage and plasmid DNAconstructs expressing combinatorial VELs representing HIV-1gp120 V3-loop-derived mouse H-2Dd-restricted immunodominantCTL epitope L (311RGPGRAFVTI320), designated LWT. The generalscheme for immunogen construction is presented in Fig. 1. In orderto introduce mutations LWT, degenerate oligonucleotides (oligos)were designed (Table S1) encoding a library of epitope variants withstructural composition RGPGXAXXXX or XGXGXAXVXI, where X isany of 20 natural amino acids. Thus, each variant epitope has ran-dom amino acid substitutions (mutations) at five defined positionswithin the LWT. Although the theoretical complexity of such libraryis 3.2 × 106 individual members, we decided to test immunogenicproperties of VELs with lower complexities first.

We chose Ig heavy-chain variable (VH) domain as efficientmolecular context for CD8+ T-cell epitope expression, as we havereported previously (Manoutcharian et al., 1999). First, a DNAfragment encoding wild-type VH domain was generated by PCRassembly using a panel of oligos (Fig. 1A and Table S1). Then, modi-

fied VH-encoding DNAs were constructed by replacing oligo codingfor Ig-VH complementarity-determining-region 3 (HCDR3) withan oligo encoding LWT or the mixture of above-mentioned VEL-encoding degenerate oligos, respectively (Fig. 1A). We cloned theDNA fragments into eukaryotic expression vector VHExpress, con-

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aining a genomic DNA encoding Ig Fc portion (Persic et al., 1997)Fig. 1B and C), resulting in plasmid DNA constructs VVH, VHPL andHBL that express control wild-type VH, LWT and LWT-based VEL,espectively. The VHBL epitope library had a complexity of about0,000 individual members, each of them representing a uniqueutated variant of LWT.In order to express the epitopes in the context of recombinant

13 bacteriophages, two DNA fragments corresponding to HCDR3nd encoding LWT and VEL, respectively, were amplified by PCR andloned into pG8SAET phagemid vector (Manoutcharian et al., 2004)hat allows the expression of epitopes at high copy numbers aseptides fused to phage cpVIII. Thus, phage display VEL-expressing

ibrary (BFL) with the complexity of about 12,500 individual cloness well as a recombinant phage expressing nominal LWT (ELN) wereenerated. To check the correct cloning and to generate antigens for-cell proliferation assay, 20 phage clones were isolated randomlyrom the BFL and the deduced amino acid sequences of corre-ponding peptides/epitopes were determined by DNA sequencingTable 1). Unexpectedly, while an equimolar mixture of two DNAragments encoding epitope variants with two structures (RGPGX-XXXX and XGXGXAXVXI) were used in the cloning step, only 2 outf 20 isolated clones had RGPGXAXXXX epitope composition andhe rest had XGXGXAXVXI structure. At mutated particular aminocid positions in 20 epitope variants, 9–12 different amino acidsere detected, indicating an acceptable epitope diversity of bothFL and VHBL combinatorial libraries (Table 1). Notably, the iso-

ated phage clones are not present in BFL, since they were isolatedefore the amplification of BFL and, the chance for the presence ofhese clones in VHBL is obviously negligible. Three additional phageibraries BFL4, BFL3 and BFL2 expressing VELs with 4, 3 or 2 ran-omized amino acid positions corresponding to epitope structuresGPGRAXXXX, RGPGRAFXXX and RGPXRXFVTI, respectively, wereonstructed using degenerate oligos (Table S1) as described above.

.2. Cellular immune responses induced by VELs

To evaluate the ability of BFL phage-displayed epitope librarynd VHBL DNA construct to elicit immune responses to CTL epi-ope variants carrying multiple amino acid substitutions, groupsf 3–5 BALB/c mice were immunized by single injection withFL and VHBL, respectively. The control animals were immunizedith non-related phage GK1 and with plasmid DNA of control

ector VVH. 15 days after the immunization, splenocytes wererepared, stimulated in vitro with 20 phage clones or syntheticeptides representing L epitope variants, and the breadth (num-er of responding epitope variants) and the magnitude of T-cellroliferative responses were measured.

While splenocytes from the BFL-immunized mice exhibitedtrong responses to L-derived epitope variants with SIs rangingrom 30 to 87, the spleen cells from GK1-treated mice had back-round SI levels (SIs 10–22) (Fig. 2A), indicating the induction ofEL- and epitope-specific responses. At least 11 L epitope variantsere recognized by spleen cells from BFL-immunized mice, while

ther variants induced responses similar to those obtained when1 phage was used as control antigen (Fig. 2A). The recall responsesgainst individual epitope variants, selected at random, were highlyeproducible and, interestingly, several epitope variants resultedore potent antigens than LWT epitope in lymphoproliferation

ssays (Fig. 2A and B). Similar results were obtained when spleenells from mice immunized with VHBL DNA construct were testedn proliferation assays, although the general magnitudes of immune

esponses were lower (Fig. 2B). Background levels of activationere obtained when spleen cells from mice immunized with con-

rol phage GK1 and plasmid DNA of VVH vector were stimulatedy the same panel of antigens (Fig. 2A and B). Worth noting, thathe epitope variants L5–7, L1 and L9 that induced most potent

munology 47 (2009) 270–282

T-cell proliferation with cells from BFL-immunized mice, werealso the best inducers of proliferation of splenocytes isolated fromVHBL-immunized mice, indicating that the activated T cells areepitope-specific and the immune responses were independent ofthe molecular context or the route of application of VEL immuno-gens.

When 10 epitope variants were tested as synthetic peptides, 7of them that were good cell stimulators as phage antigens, acti-vated the cells from both BFL and VHBL-immunized mice and, onlytwo variants (L20 and L12), that were negative as phage antigens inprevious assays, activated T cells when used as synthetic peptides(Fig. 2C). Again, the control GK1- and VVH-immunized mice werenot able to generate cross-reactive responses and, LWT-expressingELN phage and VHPL DNA construct induced immune responsesthat only weakly recognized a few epitope variants, including LWTbearing synthetic peptide PNL. These data indicate the superior-ity of VEL-based immunogens over immunogens carrying defined(non-mutated) epitopes, in their capacity of inducing broad andpotent immune responses. Also, the theoretical affinities of inter-actions between peptides and MHC molecules were calculatedshowing no significant correlation between the levels of affinitiesand the magnitude of T-cell responses (Fig. S1).

We determined next whether decreasing the complexity of VELswould affect the breadth of immune responses. Mice were immu-nized with phage-displayed BFL4, BFL3 and BFL2 libraries eachexpressing VELs with mutations at 4, 3 or 2 amino acid positions,respectively. A clear reduction of the immunogen potency and thenumber of variant epitopes recognized by spleen cells from animalsreceiving VELs bearing epitope libraries of lower complexities wereobserved compared to the mice immunized with BFL (Fig. 2D andE). The same patterns of epitope recognition were observed whereboth phage and synthetic peptides were used as antigens.

3.3. Phenotypic analysis of activated lymphocytes

To determine the subpopulations of proliferating T cells and totest whether the immunization with VELs induced epitope-specificactivation of CD8+ IFN-�+ cells, since the production of IFN-� byactivated CD8+ T cells is a typical phenotype of CTLs, we usedflow cytometry and intracellular cytokine staining (ICS) (Fig. 3A).The pooled splenocytes from each group were analyzed by FACSeither after 6 h of stimulation (ex vivo cells) or upon 3 or 6 days ofincubation with phage-displayed LWT (ELN), non-related epitopeB1, two variant epitopes and BFL, respectively. As shown, whileex vivo the majority of CD3+ T cells from VHBL-immunized miceshowed CD4+ phenotype (40–50%), after stimulation the percent-age of CD8+ T cells rose by 10% constituting the majority of cellpopulation, exempt when control B1 antigen was used as stim-ulator leading only to slight increase in the number of CD8+ Tcells (Fig. 3B). Similar profiles of T-cell activation accompanied bydramatic changes in CD4/CD8 cells balance after stimulation wereobserved with cells from BFL-immunized mice, although this timewe detected only small increase in splenocytes stimulated withBFL (Fig. 3C). The latter was not unexpected, considering that M13phage particles are strong immunogens capable of recruiting CD4+helper cells.

Next, intracellular IFN-� expression in CD8+ lymphocytes stim-ulated with a panel of phage-displayed variant epitopes wasanalyzed, and positive cells were enumerated. Cells isolated frommice immunized with VHBL or BFL contained significant propor-tion of IFN-�-producing epitope-specific cells (7–12% and 6–10%,

respectively) as shown in Fig. 3D and E. Stimulation of splenocytesfrom DNA- and phage-immunized mice with 5 and 6 epitopes,respectively, out of 10 variant epitopes tested, led to activationof IFN-�+ cells, while incubation of spleen cells with the rest ofthe variants resulted in background levels of IFN-�-producing cells

C. Pedroza-Roldan et al. / Molecular Immunology 47 (2009) 270–282 275

Fig. 2. Immunization with VELs induced potent cellular responses against multiple epitope variants. BALB/c mice (n = 3) were immunized with 1 × 1011 CFU M13 phageparticles bearing BFL or control GK1 clone by i.p. injection (A), or were genetically immunized i.d. with 75 �g of VHBL or VVH control plasmid DNA (B). Other groups of micew 2, or Em or con( ± SDo

oTec

ere immunized with BFL, ELN or GK1 (C), with phage libraries BFL, BFL4, BFL3, BFLice were stimulated (3 days) with a set of phage-displayed epitope variants, LWT

10 �M) (C) and, stimulation indexes (SIs) were calculated. Data shown are the meanut.

f 5% (obtained after stimulation with control B1 phage antigen).he stimulation of splenocytes from mice immunized with LWT-xpressing DNA construct VHPL and ELN phage clone, as well asells from control DNA VVH and GK1 phage-immunized mice, with

LN and GK1 phage (D and E). After 15 days, the splenocytes isolated from individualtrol B1 phage (1 × 1010 CFU/well) (A, B, D and E) or (5 days) with synthetic peptides(n = 3). Three (A, B, and C) and two (D and E) independent experiments were carried

the same set of variant epitopes, gave the cell activations belowthe background levels (Fig. 3D and E). Interestingly, as in cell pro-liferation assays, several variant epitopes resulted in better cellstimulators than LWT epitope and, 3 variants (L20, L15 and L4) were

276 C. Pedroza-Roldan et al. / Molecular Immunology 47 (2009) 270–282

Fig. 3. Phenotypic analysis of activated T cells by FACS. Mice were immunized either with plasmid DNA (B, D and F) or phage particles (C, E and G) as described in the legendto Fig. 2. On day 15 after the priming, splenocytes were collected, pooled and intracellular stained (ICS) for IFN-� after 3 (D and E) and 5 days (F and G) of stimulation with apanel of epitope variants either as phages (B–E) or as synthetic peptides (F and G). The CD4 and CD8 positive T-cell populations of proliferating lymphocytes were determinedby FACS after 6 h (B and C) of stimulation or 3 (B) and 6 days (C). As control immunogens VVH vector DNA and GK1 phage clone were used and, B1 phage and syntheticpeptide PB1 served as negative control antigens. (A) Representative plots show the percentages of epitope-specific CD8+ IFN-� cells isolated from VHBL DNA-immunizedmice in the upper right quadrant.

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ble to stimulate cells from mice immunized with both VHBL andFL. We prepared also synthetic peptides corresponding to above-escribed variant epitopes and, used them in similar FACS assays.gain, cells from DNA- and phage-immunized mice were activated

or IFN-� production by 6 and 5 peptide epitopes, respectively,ut of 10 variant peptide epitopes tested, although in this case theagnitudes of responses were lower in general (Fig. 3F and G).

.4. Induction of memory responses and immune interference

We determined next whether the immunization with VELsould induce long-lasting memory T-cell responses. Centralemory (TCM) and effector memory (TEM) CD8 T cells can be distin-

uished by high and low levels of CD62L expression, respectively.wo groups of mice were primed by immunization with BFL or con-rol phage GK1. 7 months afterwards, spleen cells were analyzedy FACS (Fig. 4A) and cell proliferation assays. While a typical pro-ortion of TCM/TEM is about 4 to 1 in non-stimulated splenocytesunstimulated cells) from immunized or intact mice, the proportionf TEM increased up to 40% of CD44+ cells from BFL-immunized micefter stimulation with ELN, L9 and L6 (Fig. 4B). At the same time, noubstantial changes were observed in TCM/TEM cell balance upontimulation of the cells with control B1 phage or when splenocytesrom GK1-immunized mice were stimulated with the same anti-ens (Fig. 4B). These data indicate that the activation of TEM cells ispitope-specific.

In order to analyze the breadth and the potency of immuneesponses, splenocytes were tested in antigen recall studies usingset of epitope variants. The splenocytes from mice primed withFL and VHBL were efficiently proliferating after stimulation withand 5 variant epitopes, respectively, out of 10 variant epitopes

ested (Fig. 4C and D). Strikingly, the magnitude and the breadthf immune responses in mice primed with VHBL and tested either5 days (Fig. 2B) or 7 months later, were very similar and, in BFL-rimed mice (Fig. 2A) only about 30% decline of immune responseagnitude was observed, where the results from these time pointsere compared. Testing of the same set of synthetic peptides with

he cells from VHBL-primed mice revealed that 7 out of 10, were stillell stimulators (Fig. 4E), albeit the magnitude of responses wereower compared to data from mice tested 15 days after primingFig. 2C).

Finally, we decided to recreate a situation when an individualaccinated with immunogens containing defined epitopes (almostll current vaccine development approaches are based on suchmmunogens) is infected with AVPs carrying diverse pool of relatednd rapidly evolving epitopes. The mice were primed with VVHr VHPL, boosted by single immunization with VHBL 8 monthsater and, recall responses against a panel of variant epitopes werenalyzed. While strong responses against the majority of variantpitopes, tested either as phage (Fig. 5A) or synthetic peptidesFig. 5B), were detected in splenocytes from VVH-primed andHBL-boosted mice, a significant reduction in the magnitude andreadth of responses were observed after incubation of the same setf epitopes with spleen cells from VHPL-primed and VHBL-boostedice (P < 0.02) (Fig. 5A and B). Interestingly, strong recognition of

WT epitope, responsible for immune interference, was maintainedntact when we tested LWT in the form of both phage (ELN) ands synthetic peptide (PNL) (Fig. 5A and B). Strikingly, the observedhenomenon of immune interference in LWT bearing VHPL-primedice, also known as original antigenic sin (OAS) or deceptive

mprinting (Tobin et al., 2008; Klenerman and Zinkernagel, 1998),

as even more pronounced in mice primed with VHPL and tested

2 months later. This time, we primed the mice with VHPL and,n additional group of animals were immunized by single DNAnjection with VHBL. After 12 months, all animals were boosted

ith VHBL and recall responses were measured. As expected, while

munology 47 (2009) 270–282 277

most potent responses were obtained in spleen cells from VHBL-primed VHBL-boosted mice: 13 and 7 variant epitopes out of 16phage and 10 synthetic peptide epitopes tested, respectively, werepositive more than 3-fold reduction of the magnitude of responsesagainst the same variant epitopes was observed in spleen cells fromVHPL-primed mice (P < 0.018) (Fig. 5C). Intermediate values of cellproliferation were obtained when splenocytes from VVH-primedmice were tested using synthetic peptides (Fig. 5C). Collectively,these data indicate that priming with defined antigen/epitopeinduced strong inhibition of immune responses potentially capa-ble of recognizing variant epitope(s) through OAS and, importantly,the level of maturation of T-cell immune responses depending ontiming between priming and boosting (8 months vs. 12 months)with the immunogen may enhance this effect.

4. Discussion

The development of vaccines against AVPs and diseases rep-resents an unprecedented challenge and, classical approaches ofvaccine design, although effective mostly for antigenically non-variable pathogens, were not and, most probably, will not besuccessful against AVPs and cancer. For example, 25 years afterthe discovery of human immunodeficiency virus (HIV), despitethe unprecedented breadth and rapid pace of scientific accom-plishments in the field of HIV research, there is still no effectivevaccine (Walker and Burton, 2008; Buchbinder et al., 2008). Thus,non-classical approaches and even conceptually new thinking isreadily required for the development of novel immunogens capa-ble of inducing protective immune responses against cancer, HIVand other AVPs. While it is clear that the antigenic diversity is themain obstacle to the development of these vaccines, technically,the problem is just to find a way to incorporate this diversity intothe vaccine immunogens. Hence, we are proposing a simple solu-tion to this challenging task and have developed an immunogenconstruction platform that specially designed to deal with AVPsand diseases. This approach is completely new and, the results pre-sented here strongly suggest that our approach is feasible and mayconstitute a new avenue to target antigenically variable diseases ingeneral.

The aim of this proof-of-concept study was to test the capacityof the immunogens based on novel VEL vaccine design approach toelicit broad immune responses capable of recognizing mutated epi-tope variants using as a model HIV immunodominant CTL epitope.We clearly showed that the VELs used both as DNA constructs oras recombinant M13 phage, induced a potentially large repertoireof IFN-� producing CD8+ T cells recognizing a diverse set of CTLepitope variants carrying mutated (substituted) amino acids at 5out of 10 amino acids positions of the epitope in each epitope vari-ant. The majority of 20 epitope variants randomly selected fromVEL of about 104 individual members were recognized by CD8 Tcells indicating that we may have diverse pool of about 5000 dif-ferent TCR-bearing T cells in immunized mice. Most remarkably, weshowed the induction of long-lasting (up to 1 year) memory T cellsby a single immunization with VELs. In addition, we showed in vivoimmune interference consisting in inability of mice primed withwild-type epitope to generate efficient T-cell responses againstvariant epitopes after boosting with VELs. The latter, to our knowl-edge, is the first demonstration of OAS in terms of large panelof epitope variants and time frame. We used M13 recombinantphages, displaying epitopes/peptides in dense repetitive arrays onphage converting them in strong immunogens without the need for

any adjuvant, as carriers. We have reported such phages as effectivecarriers for practical vaccines for the first time (Morales et al., 2008).Despite this, and considering that we carried out genetic immu-nization by simple DNA injection, the application of other modernvaccine delivery systems, such as in vivo DNA electroporation and

278 C. Pedroza-Roldan et al. / Molecular Immunology 47 (2009) 270–282

Fig. 4. Induction of memory responses by VELs. The groups of Balb/c mice (n = 5) were immunized with BFL (B and C) or VHBL (D and E) and, the control groups received singleinjection with GK1 phage and VVH vector DNA. Seven months later, pooled spleen cells either unstimulated or stimulated with phage epitopes were examined by FACS (Aand B) by measuring changes in the expression of CD62L. Representative plots show the percentages of epitope-specific CD8+CD62L+ cells in the upper right quadrant uponstimulation with antigens (A), which are also presented graphically (B). The splenocytes isolated from individual mice were stimulated with the panel of variant epitopesas phage (C and D) or as synthetic peptides (E), including antigen controls, and their proliferative capacities (SIs) were measured as described in the legend to Fig. 2. Datashown are the mean ± SD (n = 5). Two independent assays were carried out.

C. Pedroza-Roldan et al. / Molecular Immunology 47 (2009) 270–282 279

Fig. 5. Induction of immune interference (original antigenic sin). Groups of mice (3 per group) were primed by single DNA injection with VVH, VHPL or VHBL. 8 monthslater, the mice primed with VVH and VHPL were boosted by immunization with VHBL DNA construct and pooled spleen cells proliferations, after stimulation with variantp ost (Aa nths ap ll prolw

vpTnb

hage epitopes (A) or synthetic peptides (B), were measured on day 15 after the bodded in other study, where three groups of mice were boosted with VHBL 12 moanels each point represents an individual epitope variant used as antigen in T-ceere considered statistically significant. Two independent assays were conducted.

iral vectors along with the addition of molecular adjuvants andrime/boost regimes may result in more robust immune responses.he VELs could be easily modified, for example by reducing theumber of mutated amino acid positions, as shown in this study, ory applying VELs with lower complexities, to more closely resem-

and B) as described in the legend to Fig. 2. A group of mice primed with VHBL wasfter the priming and, spleen cell were used in proliferation assays (C). In the rightiferation assays. The P values were calculated using two tailed t test and a P < 0.05

ble particular antigenic profiles and dynamics of the evolution ofpathogen’s antigenic profile and to avoid possible harmful autoim-mune responses. Likewise, several VELs derived from different T-and B-cell epitopes, including both immunodominant and subdom-inant epitopes, can be combined in a single mixture allowing a

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arger coverage of epitope profiles, thus, reducing the probabili-ies of immune escape. We showed also the generation of sera withroad HIV-1-neutralizing activity against primary viral isolates by

mmunizing mice both with plasmid DNA construct (VHBL) andecombinant M13 phage (BFL) carrying HIV-1 CTL (which is also anmmunodominant B-cell epitope) LWT epitope-derived VELs (ournpublished data).

There are very few reports describing recognition of HIV-1ariant epitopes by vaccine-induced T cells such a study describ-ng recognition of variant HIV-1 epitopes by spleen cells of micemmunized with multi-epitope DNA plasmid vaccine expressingonserved CTL epitopes intended to induce broad T-cell responsesMcKinney et al., 2004). However, most variant epitopes con-ained single amino acid substitution or substitutions at HLAinding non-anchor sites and, importantly, only epitopes carryingLA peptide-binding motif were included in that study. Despite

his, less than 50% of variants, with 3 or 4 amino acid changesere recognized and, in general, recognition rates decreased as

he number of substitutions increased (McKinney et al., 2004).herefore, the recognition and/or cross-recognition of our dra-atically mutated (including HLA anchor sites) and randomly

elected epitope variants by VEL-induced T cells are impressivend important data. These results indicate that generating a largeool of diverse CD8+ T cells will recognize also rapidly mutat-

ng epitopes of HIV-1, thus providing bases for rational designf vaccines. The above-mentioned DNA vaccine design has theame drawbacks as previously discussed approaches (see Section) that use conserved or consensus pathogen-derived sequencesor immunogen construction. Thus, it is not surprising that above-

entioned DNA vaccine was not immunogenic, as was shown in aecent human clinical study (Gorse et al., 2008). Similarly, a multi-pitope peptide (MEP) vaccine was not able to elicit HIV-specificmmunity in humans even with the application of recombinantranulocyte-macrophage colony stimulating factor (GM-CSF) pro-ein as a coadjuvant (Spearman et al., 2009).

There is a huge number of potential epitopes in viral, tumornd model systems that are encoded by non-primary open read-ng frame (ORF) sequences or derived from other non-traditionalources, such as transcriptional/translational mechanisms or splic-ng events, collectively referred as epitopes derived from defectiveibosomal products (DRiPs) (Mayrand and Green, 1998; Yewdellnd Nicchitta, 2006). Undoubtedly, the genetic variability of AVPsnd genomic instability of cancel cells lead to the generation ofhese “unexpected” epitopes that are probably the targets of a sig-ificant portion of cellular immune responses. Therefore, the poolf targeted epitopes and corresponding TCRs in cancer and in infec-ions with AVPs is huge, although many of these phenomena wereot studied systematically and are not considered in current vac-ine efforts. Hence, considering that both the TCR repertoire andVP- and cancer-derived epitopes are of random origin, as shown

n recent studies evaluating HIV-infected identical twins (Yang etl., 2005; Yu et al., 2007), the VELs carrying random mutations areerfectly suited as immunogens capable of dealing with antigenicariability. In other words, the new strategy proposed here attackshe disease using their own weapons: rapid and random mutationates.

Importantly, there is a potential danger in possible massivepplication of current HIV candidate vaccines, which is also trueor other AVPs, since they may reduce the pool of naïve T cells byocusing the immune response on few immunodominant epitopesncluded in vaccines, which may lead to much faster development

f AIDS in vaccinated persons. The antigenic variability not onlynduces escape mutants, but also may lead to heterologous immu-ity (Brehm et al., 2002; Cornberg et al., 2006) and OAS (Tobint al., 2008; Klenerman and Zinkernagel, 1998): two immuno-ogical phenomena, representing serious obstacles for current

munology 47 (2009) 270–282

vaccine development, which might point out possible mechanis-tic explanations for vaccines failure. Indeed, while vaccine-elicitedepitope-specific CD8+ T cells have a clonal diversity comparable tothose cells generated in response to SHIV challenge in monkeys,the clonal TCR diversities are restricted or clonally focused in bothcases, as shown in a recent study (Sen et al., 2008). The epitopeescape may be also a consequence of heterologous viral challengeresulting in a narrow oligoclonal TCR repertoire (Cornberg et al.,2006). Interestingly, some immune interference caused by OAScan be avoided by simultaneous delivery of mutant and originalCTL epitopes to the immune system (Singh et al., 2002). Impor-tantly, in a recent follow-up study of the failed first HIV vaccineefficacy trial conducted by VaxGen, significantly higher activationof both CD4 and CD8 T cells was found in vaccine responders whosubsequently became infected with HIV (Jones et al., 2009). Thecell-mediated adenovirus serotype 5 (Ad5)-based MRKAd5 HIV-1 gag/pol/nef recent vaccine trial conducted by Merck, althoughdemonstrated that the vaccine was highly immunogenic for induc-ing HIV-specific CD8+ T cells (McElrath et al., 2008), showed noevidence or future likelihood for vaccine efficacy and, the numberof infected persons was even higher among vaccinated individualsthan in the control group (Buchbinder et al., 2008). Surprisingly,above-mentioned HIV vaccine candidates were generated withouteven addressing the issue of antigenic variability, which is truealso for other AVPs, and consequently, were not tested in theircapacity to induce immune responses able to recognize variant(mutated) epitopes. Therefore, we think that there is a possibilitythat the recent failure of these trials is a direct result of above-mentioned OAS and/or heterologous immunity, rather than othersuggested explanations, such as preferential infection of activatedT cells (either HIV-specific or not) or the influence of pre-existingAd5 neutralizing Abs. A possible proof for our suggestion came froma recent study showing that cats vaccinated with Env or Gag pro-tein of feline immunodeficiency virus (FIV) and challenged withhomologous FIV strain, developed viral RNA earlier as compared tocontrol animals (Klonjkowski et al., 2009).

In conclusion, the vaccines based on defined antigens carryinglimited number of epitopes, most probably, may hamper or inhibitthe activation of naive T cells upon encounter with pathogen bear-ing slightly different antigen/epitope variants. We are convincedthat the reason for the failure of HIV/AIDS vaccines and the vac-cines against other AVPs is the incorrect design of the vaccinesthemselves, which are carrying defined antigenic profiles, whetherin the form of whole pathogens, genes, proteins or epitopes, thatlargely differ from antigenic/immunogenic compositions and pro-files of targeted pathogens and disease conditions, rather than thelack of immunogenicity or the vaccine potency. Furthermore, allabove-mentioned data along with the demonstration of immuneinterference at molecular level in the present study are clearlyindicating that: current vaccine efforts against AVPs are basedon incorrect immunogen design concepts; such “bad vaccines”represent serious danger for the people in the case of massive appli-cation; while with HIV, HCV, malaria or cancer vaccines there isno immediate threat due to obvious reason (there are no practicalvaccine candidates yet), the application of flu vaccines, includinginfluenza A H1N1, might be harmful for the large portion of popu-lation.

We believe that VEL-based immunogens will induce protectiveimmune responses against AVPs and cancer, when used as molec-ular vaccine components in both prophylactic and therapeuticsettings. Our vaccine concept closely resembles the natural infec-

tions with AVPs and diseases in respect to interactions betweenimmune system and rapidly evolving epitopes. The vaccines againstAVPs should not only match to pathogen’s antigenic composi-tion, but, most importantly, should somehow reflect the dynamicsof pathogen’s evolution in vivo at epitope level (Fischer et al.,

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007). Hence, the VEL concept complains with these require-ents, may prevent or reduce the likelihood of epitope escape

nd, therefore, represents a realistic alternative to existing vaccinepproaches targeting AVPs and cancer. The reason of failure of vac-ines against AVPs is not the general lack of immune responses,hich are present in majority of cases, but the progressive deficits

n T-cell repertoires caused by evolving antigenic diversity lead-ng to activation of T cells that recognize non-protective epitopesnd/or are unable to recognize new epitope variants. Our hypoth-sis is that simultaneous presentation of thousands of epitopeariants to immune system will induce the activation of broadange of T cells, both CTL and Th, capable of recognizing both theathogen’s epitopes present at the time of experimental or natu-al pathogen challenge and the variants of these epitopes that willppear rapidly upon infection. Therefore, the competitive bindingf cognate peptides to MHC could be beneficial for the inductionnd maturation of protective immune responses, instead causingarmful autoimmune effects. The VELs, particularly representing

mmunodominant epitopes, may serve as powerful vaccine com-onents, since having the capacity to alter and activate diverseCR pool, they will maintain T-cell immunodominance hierarchieshich is a fundamental to the development of vaccines based on

ellular responses. Indeed, the primary determinants of immun-dominance are independent of TCR repertoire composition andiversity, as was recently shown in a mouse influenza infectionodel (Kedzierska et al., 2008). The sequencing of TCR V� clonesill permit us to define whether the induced TCR repertoire is

ligoclonal or polyclonal, which in turn, could clarify whether theecognition of epitope variants is the result of cross-recognition byligoclonal T cells or the variants are recognized by large pool ofiverse TCR-bearing T cells, respectively.

In summary, we have shown the feasibility of the generation ofmmunogens, based on incorporation of a large number of epitopeariants in a single composition, capable of inducing a broad rangef epitope variant-specific T-cell responses. The proposed conceptf immunogen design may serve as a general technological plat-orm for the design of molecular vaccines against pathogens withigh levels of antigenic variability and cancer.

cknowledgments

We thank the support of: Maria Elena Munguia for technicalssistance, Angel G. Diaz Sanchez for determining theorical Kdf the epitopes and Universidad Nacional Autónoma de México,nstituto de Investigaciones Biomédicas, Programa de Doctoradon Ciencias Biomédicas and Dirección General de Estudios deosgrado. Cesar Pedroza-Roldan is recipient of a doctoral scholar-hip from CONACYT (195086). We appreciate the training supporteceived by C. Charles-Nino from Posgrado en Ciencias Biológicas.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.molimm.2009.09.024.

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