Original Articles

P5 HER2neu-derived peptide conjugated to liposomes containingMPL adjuvant as an effective prophylactic vaccine formulation forbreast cancerSheida Shariat a Ali Badiee b Seyed Amir Jalali c Mercedeh Mansourian b Mona Yazdani bSeyed Alireza Mortazavi a Mahmoud Reza Jaafari da Department of Pharmaceutics School of Pharmacy Shahid Beheshti University of Medical Sciences Tehran Iranb Nanotechnology Research Center School of Pharmacy Mashhad University of Medical Sciences Mashhad Iranc Department of Immunology Medical School Shahid Beheshti University of Medical Sciences Tehran Irand Biotechnology Research Center Nanotechnology Research Center School of Pharmacy Mashhad University of Medical Sciences Mashhad Iran

A R T I C L E I N F O

Article historyReceived 19 June 2014Received in revised form 25 August 2014Accepted 9 September 2014

KeywordsMPL-LiposomePeptide vaccineCTL epitopeHER2neu peptideBreast cancer

A B S T R A C T

Vaccines containing synthetic peptides derived from tumor-associated antigens (TAA) can elicit potentcytotoxic T lymphocyte (CTL) response if they are formulated in an optimal vaccine delivery system Theaim of this study was to develop a simple and effective lipid-based vaccine delivery system using P5 HER2neu-derived peptide conjugated to Maleimide-PEG2000-DSPE The conjugated lipid was then incorporatedinto liposomes composed of DMPCDMPGCholDOPE containing Monophosphoryl lipid A (MPL) (Lip-DOPE-P5-MPL) Different liposome formulations were prepared and characterized for their physicochemicalproperties To evaluate anti-tumoral efficacy BALBc mice were immunized subcutaneously 3 times intwo-week intervals and the generated immune response was studied The results demonstrated that Lip-DOPE-P5-MPL induced a significantly higher IFN-γ production by CD8+ T cells intracellularly whichrepresents higher CTL response in comparison with other control formulations CTL response inducedby this formulation caused the lowest tumor size and the longest survival time in a mice model of TUBOtumor The encouraging results achieved by Lip-DOPE-P5-MPL formulation could make it a promisingcandidate in developing effective vaccines against Her2 positive breast cancers

copy 2014 Elsevier Ireland Ltd All rights reserved

Introduction

Despite many decades of research on the cancer treatment canceris still a major cause of death Chemotherapy radiotherapy andsurgery are current treatments for cancers however chemothera-py destroys cells indiscriminately and radiotherapy and surgery arenot able to prevent metastases Due to the disadvantages of currenttreatments for cancers tumor immunotherapy has been paid at-tention during two past decades [1] Since humoral immunity hasa low potential to eliminate solid tumors individually induction ofan effective cell-mediated immunity based on the activation of cy-totoxic T lymphocytes (CTLs) namely CD8+ T cells is aimed in cancerimmunotherapy [23]

Vaccines containing synthetic peptides derived from tumor-associated antigens (TAA) can elicit potent CTL response if theyare formulated optimally Ag-presenting cells (APCs) mainly den-dritic cells (DCs) present peptide antigens to T cells (CD4+ and CD8+)via MHC molecules and initiate immune responses to infectious

diseases and tumors [45] Exogenous peptide antigens which aretaken up by DCs pass the endocytic pathway and they are gener-ally presented to CD4+ T cells on MHC class II molecules whereasendogenous antigens enter into the cytosol load onto MHC class Imolecules in the endoplasmic reticulum and are finally presentedto CD8+ CTLs [6] Therefore efficient delivery of TAAs to DCsendosomal escape of antigens to the cytosol and activation of CTLsvia MHC class I presentation are crucial to induce an effectiveimmune response leading to tumor regression

Nanoparticle delivery systems carrying antigens have the po-tential for achieving all the above mentioned goals Liposomes canoffer several advantages over other particulate systems Basicallyliposomes are safe and well-tolerated carriers They are also com-pletely biodegradable and versatile to be formulated with differentlipid constituents all types of peptide antigens and adjuvants toinduce a robust cell-mediated immunity [78]

Adjuvants in liposomal vaccine formulations can enhance andprolong immune responses [9] Among different adjuvantsMonophosphoryl lipid A (MPL) has been used frequently as an ef-ficient adjuvant in liposomal vaccines MPL has shown adjuvantactivity in both cellular and humoral immunity [10] MPL is a non-toxic derivative from LPS or endotoxin that drives immunity

Corresponding author Tel +98 511 8823255 Fax +98 511 8823251E-mail address Jafarimrmumsacir (MR Jaafari)

httpdxdoiorg101016jcanlet2014090160304-3835copy 2014 Elsevier Ireland Ltd All rights reserved

Cancer Letters 355 (2014) 54ndash60

Contents lists available at ScienceDirect

Cancer Letters

journal homepage wwwelseviercom locate canlet

responses via TLR4 stimulation [1112] FDA approved MPL as a safeadjuvant for human vaccines [13]

Through developing tumor-specific peptide vaccines various TAAshave been targeted for cancer immunotherapy As a TAA HER2neu protein has provided an opportunity to develop breast cancervaccines HER2neu is a 185 kDa transmembrane glycoprotein andmember of the epidermal growth factor receptor family over-expressed in 20ndash40 of primary breast cancers [1415]

In our previous study four peptides containing MHC class I re-stricted multi-epitope from rat HER2neu protein were designed byin silico analysis and the effectiveness of these peptides was evalu-ated by administration to BALBc mice As results showed that twoof these peptides (p5 and p435) were effective in inducing CTL re-sponses it was hypothesized that encapsulating P5 or P435 in lipidcarriers may enhance CTL immune responses more than peptidesalone Encapsulating peptides in LPD (liposome-polycation-DNA)nanoparticles included DOTAP as a cationic lipid and CpG ODN asan immune-stimulatory adjuvant confirmed the hypothesis [16]However LPD is a complex carrier and PS-type CpG ODN at highdose may elicit systemic toxicity [17]

For these reasons in the present study we utilized liposomescomposed of DMPCDMPGCholDOPE containing MPL for efficient-ly introducing P5 peptide to cytosol of APCs and generating a strongCTL response In our earlier challenging study we developed an op-timized procedure for encapsulating P5 peptide in the inner cavityof liposomes by passive loading [18] As encapsulation efficiencywas low in this study P5 peptide (ELAAWCRWGFLLALLPPGIAGGGC)was covalently conjugated to Maleimide-PEG2000-DSPE to improvepeptide incorporation into liposomes The effectiveness of lipo-somal formulation of P5 peptide in the induction of CTL responsewas evaluated in BALBc mice and in TUBO in vivo tumor mice modelwhich overexpresses the HER2neu oncogene

Materials and methods

Materials

Peptide P5 (ELAAWCRWGFLLALLPPGIAGGGC purity gt 95) was synthesized byChinaPeptides Co (Shanghai China) Dimyristoylphosphatidylcholine (DMPC)dimyristoylphosphoglycerol (DMPG) dioleoylphosphatidylethanolamine (DOPE) anddistearoylphosphoethanolamine-N-[maleimide(polyethylene glycol)-2000](Maleimide-PEG2000-DSPE) were purchased from Avanti Polar Lipid (Alabaster USA)Cholesterol and Monophosphoryl lipid A from Salmonella enterica (MPL) were pur-chased from Sigma-Aldrich (Steinheim Germany) CytofixCytopermTM Plus PMAionomycin cocktail anti-CD8a-PE-cy5 anti CD4-PE-cy5 anti-IFN-γ- FITC and anti-IL-4-PE antibodies were purchased from BD Biosciences (San Diego USA) All othersolvents and reagents were used as chemical grade

Animal and cell lines

Four to six week old female BALBc mice were purchased from Pasteur Insti-tute (Tehran Iran) The experimental protocols were approved by the InstitutionalEthical Committee and Research Advisory Committee of Mashhad University ofMedical Sciences in accordance with animal welfare guidelines

TUBO a cloned cell line that overexpresses the rHER2neu protein was kindlyprovided by Dr Pier-Luigi Lollini (Department of Clinical and Biological SciencesUniversity of Turin Orbassano Italy) and was cultured in Dulbeccorsquos Modified EaglersquosMedium (DMEM) and supplemented with 20 fetal bovine serum (FBS) A murinecolon carcinoma cell line CT26 was purchased from Pasteur Institute (Tehran Iran)and cultured in RPMI-1640 medium supplemented with 10 FBS

Conjugation of P5 peptide to PEG2000-DSPE

P5 peptide was conjugated to Maleimide-PEG2000-DSPE through covalent bindingbetween the thiol group of cysteine residue of peptide and the pyrrole group ofmaleimide Peptide was reacted with Maleimide-PEG2000-DSPE in a molar ratio of121 (peptidemaleimide) in DMSOchloroform (11) solution at room tempera-ture for 24 h Thin layer chromatography (TLC) was used to confirm the formationof P5-PEG2000-DSPE A TLC plate (silica gel 60 F254 Merck USA) was placed in a TLCchamber containing mobile phase composed of chloroform methanol and waterat 90182 (vv) The chamber was saturated with iodine vapor to stain the TLC plateThe conjugation of peptide with PEG2000-DSPE was also ascertained indirectly by

determining unconjugated peptide fraction using HPLC KNAUER smart line HPLC(Berlin Germany) was equipped with a Nucleosil C18 5 μm 150 times 46 mm 100Adegcolumn (KENAUER) and an UV detector (KENAUER S2600) set at 220 nm The mobilephases employed were A (water + 01 TFA) and B (acetonitrile + 01 TFA) Elutionprogram was a gradient starting with 100 A and increasing to 30 B in 2 min 60B in 10 min and 90 B in 2 min The flow rate was set to 1 mlmin

Liposome preparation

Liposomes (Lip-DOPE) composed of DMPCDMPGCholDOPE at a molar ratioof 304610 were prepared using lipid film hydration method Control liposomes(Lip) were also prepared in the same molar ratio as above without using DOPE Lipidswere first dissolved in chloroform and then they were combined in sterile glass tubesThe required amount of MPL and P5-PEG2000-DSPE conjugate was added to the lipidsolutions to prepare liposomes containing P5 peptide and MPL (Lip-P5-MPL) Thelipid solutions were dried to a thin film by rotary evaporation (Heidolph Germany)under reduced pressure Films were freeze-dried (VD-800F Taitech Japan) over-night to remove the solvents completely Lipids were then hydrated in HEPES buffer(10 mM pH 72) containing 5 dextrose vortexed and bath-sonicated to dispersecompletely the lipids into the buffer The resulting multilamellar vesicles (MLVs) wereextruded using a mini extruder (Avestin Canada) to form 100 nm small unilamellarvesicles (SUVs) with a uniform size The final formulations contained 01 mgml P5peptide and 025 mgml monophosphoryl lipid A in liposome with a lipid concen-tration of 50 mM

Liposome characterization

The P5 peptide content in liposomal formulations was determined by the sameHPLC method as described in ldquoConjugation of P5 Peptide to PEG2000-DSPErdquo Lipo-some preparations were disrupted with 15 (vv) C12E10 detergent and then assayedto determine MPL content by an LAL chromogenic endpoint assay (QCL-1000 LonzaWalkersville MD) [19] The amount of total lipids was determined based on phos-pholipids by using a phosphorus assay method [20] Vesicle size polydispersity indexand zeta potential of liposomes were determined by dynamic light scattering (MalvernInstruments Malvern UK) Liposomes were stored at 4 degC under argon

Animal immunization and splenocyte collection

BALBc mice (10 per group) were immunized with different liposomal formu-lations three times at two-week intervals subcutaneously The liposome dose of 5 μmolper mouse was used for each injection Free P5 peptide (10 μgmouse) and HEPES-dextrose buffer were used as control groups

Two weeks after the last booster the mice (four per group) were sacrificed andtheir splenocytes aseptically collected to evaluate cellular immune responses

Enzyme-linked immunospot (ELISpot) assays

ELISpot assays were carried out using mouse ELISpot kits from U-cytech (UtrechtThe Netherlands) according to the manufacturerrsquos instruction Briefly one day beforemice sacrifice ELISpot 96-well plates were coated with anti-IL-4 and anti-IFN-γ an-tibodies and incubated overnight at 4 degC Splenocytes were cultured in triplicate wellsin a final volume of 200 μl with medium containing P5 peptide (10 μgml) in precoatedplates Splenocytes were incubated for 24 h at 37 degC in tissue culture incubator Whenspots appeared counting was done with Kodak 1D image analysis software (Version35 Eastman Kodak Rochester New York)

Intracellular cytokine assay via flow cytometric analysis

Splenocytes (106 cellsml) in medium containing GolgiPlugTM (1 μlml) was stimu-lated with PMAionomycin cocktail (2 μlml) for 4 h at 37 degC After stimulation 105

splenocytes were transferred into flow cytometry tubes and washed two times withstain buffer (2 FCS in PBS) Splenocytes were stained with 1 μl anti-CD8a-PE-cy5antibody and 1 μl anti CD4-PE-cy5 antibody in separate tubes for 30 min at 4 degC Thecells were washed with stain buffer and fixed using CytofixCytopermTM solutionFixed cells were washed two times with PermWashTM buffer and then stained with1 μl anti-IFN-γ- FITC antibody for 30 min at 4 degC CD4 cells were also stained with1 μl anti-IL-4-PE antibody The cells were washed with PermWashTM buffer and sus-pended in 300 μl stain buffer for flow cytometric analysis (BD FACSCaliburtrade BDBiosciences San Jose USA)

In vitro CTL assay

Two weeks after the last booster splenocytes were isolated from four mice pergroup and re-stimulated in vitro with P5 peptide (10 μgml) and recombinant IL-2(20 Uml) for 5 days After stimulation Splenocytes as effector cells were trans-ferred to U-bottomed plates in triplicate wells TUBO tumor cells in DMEM-20 wereincubated with 125 μM Calceine AM at 37 degC for one hour in the dark [21] Afterremoving the excess dye TUBO cells (2 times 104) as target cells were added to splenocytesand incubated at 37 degC for 4 hours in the dark Culture medium only and medium

55S Shariat et alCancer Letters 355 (2014) 54ndash60

containing 2 Triton X-100 were added to the wells to determine the minimum andmaximum release by target cells respectively Fluorescence in supernatants was readon a fluorimeter (FLx800 BioTek Instruments Inc USA) with excitation at 485 nmand emission at 538 nm The specific lysis was calculated as follows percentage ofspecific lysis = (release by CTLs minus minimum release by targets)(maximum releaseby targets minus minimum release by targets) CT26 cells which labeled similarly to theTUBO cells were used as negative control to prove that cytotoxic activity isspecific

In vivo tumor protection assay

The immunized mice (six per group) were challenged on day 14 post last vac-cination via subcutaneous injection in the right flank with 5 times 105 TUBO cells in 50 μlPBS buffer The tumor volume ([length times width times height] times 05) the time to reach endpoint (TTE) (from the equation of the line obtained by exponential regression of thetumor growth curve) and the percent of tumor growth delay (TGD) (based on thedifference between the median TTE of treatment group (T) and the median TTE ofthe control group (C) (TGD = [(T minus C)C] times 100]) were calculated for each mouse[2223] For ethical consideration mice were sacrificed when (a) the tumor volumewas greater than 1000 mm3 (b) the body weight loss was over 15 of initial weightor (c) the mice became lethargic or sick and unable to feed

Statistical analysis

The results were analyzed by one-way ANOVA and Tukey test to assess the sig-nificance of the differences among various formulations Mouse survival was analyzedby log-rank test (GraphPad Prism version 5 San Diego California) Results with P lt 005were considered to be statistically significant

Results

Synthesis of P5-PEG-DSPE

Formation of P5-PEG-DSPE was identified by thin layer chro-matography Disappearance of PEG-DSPE spot from the reactionmixture was confirmed by iodine vapor (Fig 1A) The extent ofunconjugated (free) peptide was determined using HPLC post re-action with PEG-DSPE and the results indicated almost completereaction between maleamide group in PEG-DSPE and thiol on P5(Fig 1B)

Physical properties of liposomal formulations

P5-PEG-DSPE was incorporated into different liposomal formu-lations to induce an effective CTL response For each formulationphysical property including liposome size polydispersity index (pdI)and zeta potential was determined as shown in Table 1

The particle size of all liposomal formulations ranged from 110to 150 nm in diameter which was desirable for vaccine formula-tions [24] Negatively charged liposomes were also homogenous andhad a uniform size with monomodal distribution (pdI lt 02)

Content of P5 peptide and MPL in liposomal formulations

Since the dose of peptide antigen and adjuvant can signifi-cantly influence the efficacy of formulations P5 peptide andMPL content were accurately determined in different liposomal for-mulations (Table 2) Based on the lipid dose of 5 μmol a similarpeptide and MPL doses per mouse were administered for eachformulation

Fig 1 Confirmation of P5 peptide conjugation to Maleimide-PEG-DSPE using TLC (A) and HPLC (B) (A) A TLC plate was placed in TLC chamber containing mobile phasecomposed of chloroform methanol and water at 90182 (vv) Disappearance of PEG-DSPE spot from the reaction mixture was confirmed by iodine vapor (B I) Standardfree peptide eluted with a retention time of ~12 minutes (B II) The extent of unconjugated (free) peptide was determined post reaction with PEG-DSPE (see method)

Table 1Vesicle size pdI and zeta potential of liposomal formulations (n = 3 mean plusmn SD)

Formulation Vesicle size(nm)

pdI Zeta potential(mV)

Lip-DOPE-MPL (DMPCDMPGCholDOPEMPL)

1263 plusmn 35 0138 plusmn 0019 minus446 plusmn 128

Lip-P5 (DMPCDMPGCholP5) 1357 plusmn 61 0173 plusmn 0014 minus417 plusmn 165Lip-P5-MPL (DMPCDMPG

CholP5MPL)1289 plusmn 63 0147 plusmn 0011 minus474 plusmn 146

Lip-DOPE-P5 (DMPCDMPGCholDOPEP5)

1423 plusmn 56 0184 plusmn 0008 minus424 plusmn 221

Lip-DOPE-P5-MPL (DMPCDMPGCholDOPEP5MPL)

1327 plusmn 93 0176 plusmn 0026 minus447 plusmn 255

56 S Shariat et alCancer Letters 355 (2014) 54ndash60

Induction of IFN-γ response by Lip-DOPE-P5-MPL formulation

The results showed that splenocytes isolated from the mice im-munized with liposomes composed of DMPCDMPGCholDOPEcontaining P5 peptide and MPL (Lip-DOPE-P5-MPL) secreted a sig-nificantly higher amount of IFN-γ than the other liposomalformulations P5 peptide alone and HEPES buffer (Fig 2A) None ofthe liposomal formulations induced considerable IL-4 response inmice (Fig 2B)

Induction of CD8+ response by Lip-DOPE-P5-MPL formulation

Flow cytometric analysis using CD8 CD4 and IFN-γ markers dem-onstrated CD8+ T lymphocytes mostly contributed to IFN-γproduction (Fig 3A and B) Moreover the results showed Lip-DOPE-P5-MPL formulation induced a significantly higher level productionof IFN-γ in CD8+ lymphocytes (higher MFI level) which repre-sented a higher number of IFN-γ producing cells in CD8+ populationor higher CTL population in comparison with other groups (Fig 3A)Flow cytometric results also showed IL-4 production in CD+4 cellsthat implies T cell-dependent humoral immunity was not inducedsignificantly in all groups (Fig 3C)

Induction of antigen-specific CTL response byLip-DOPE-P5-MPL formulation

In vitro CTL activity assay using rHER2neu-expressingTUBO tumor cells indicated immunization with Lip-DOPE-P5-MPLformulation generated significantly higher CTL response to P5antigen than other formulations (Fig 4) The CTL activity was es-tablished significantly at both various effector-to-target ratios The

Table 2Content of P5 peptide and MPL in liposomal formulations

Formulation P5 content(μgμmollipid)

MPL content(μgμmollipid)

P5 dose(μg permouse)

MPL dose(μg permouse)

Lip-DOPE-MPL ndash 491 ndash 2455Lip-P5 185 ndash 925 ndashLip-P5-MPL 189 482 945 2410Lip-DOPE-P5 196 ndash 980 ndashLip-DOPE-P5-MPL 192 508 960 2540

P5 peptide dose and MPL dose were determined based on a lipid dose of 5 μmolgiven per mouse

Fig 2 The efficacy of different liposomal formulations in inducing IFN-γ (A) and IL-4 (B) production BALBc mice (10 per group) were immunized three times at two-weekintervals with different liposomal formulations P5 peptide alone or HEPES buffer On day 14 post last booster four mice from each group were sacrificed and their splenocyteswere restimulated with P5 peptide IFN-γ and IL-4 release from splenocytes induced by different liposomal formulations was determined using ELISpot assay The data in-dicate the mean plusmn SEM (n = 4) denotes significant difference from all other formulations

Fig 3 Geometric mean fluorescence intensity (MFI) level for IFN-γ in gated CD8 (A) and CD4 (B) lymphocyte populations and MFI level for IL-4 in gated CD4s (C) Isolatedsplenocytes of immunized mice were re-stimulated in vitro with PMAionomycin and stained with CD4 CD8 IFN-γ and IL-4 markers MFI level for IFN-γ and IL-4 in gatedpopulations were determined by flow cytometric analysis The data indicate the mean plusmn SEM (n = 4) and denote significant difference from buffer and all other for-mulations respectively

57S Shariat et alCancer Letters 355 (2014) 54ndash60

cytotoxicity activity induced by Lip-DOPE-P5-MPL was specificagainst rHER2neu CTL response was not observed against rHER2neu-expressing negative CT26 cells (Fig 4)

Anti tumor effects of Lip-DOPE-P5-MPL vaccination in BALBc

Lip-DOPE-P5-MPL formulation had a superior tumor growth in-hibition in the TUBO tumor mice model (Fig 5A) The survival timewas also significantly prolonged in mice following Lip-DOPE-P5-MPL vaccination compared to the other formulations (Fig 5B) Sincenone of the mice vaccinated with Lip-DOPE-P5-MPL died during theexperiment median survival was indefinable in this group Mediansurvival time (MST) as well as TTE and TGD for each treatmentgroup are summarized in Table 3

Discussion

In the present study we attempted to enhance immunogenic-ity and adjuvanicity of P5 peptide a synthetic peptide containingCTL multi-epitope from rHER2neu protein by conjugating with

different liposomal formulations The results showed P5 peptide con-jugating with liposomes alone (Lip-P5) was incapable of inducingany immune response Although some studies reported liposomesalone could elicit CTL response against encapsulated peptide anti-gens [2526] this formulation could have been used exclusively asa carrier for antigen delivery with no potential to conduct anti-gens into the cytosol Lip-P5 formulation had a particle size of 110ndash150 nm that is needed for efficiently draining to lymph nodes whereCD8+ lymphoid DCs are present [2427]

Inclusion of a pH sensitive lipid like DOPE in the liposome struc-ture has been frequently demonstrated as an efficient strategy tointroduce antigens into MHC class I pathway [28ndash31] After endo-cytosis at the pH of the endosomal compartment the transition ofDOPE from lamellar to hexagonal phase occurs It induces the fusionof liposomes with the endosomal membrane and subsequently li-posomal antigens are released toward the cytosol [32ndash34] Howeveras results have shown inclusion of DOPE alone in liposomal for-mulation (Lip-DOPE-P5) was not sufficient to elicit an effectiveimmune response Lip-DOPE-P5 formulation might have been ableto deliver P5 antigen to MHC class I molecules successfully howeveronce the peptide antigen was presented to CD8+ lymphocytes byAPCs presence of co-stimulatory molecules on the APCs was alsorequired for activation of CD8 cells to produce CTLs [35ndash37] MPLcan induce intracellular signaling pathways leading to productionof these co-stimulatory molecules through TLR4 stimulation [35]Co-formulation of MPL and DOPE in liposomes (Lip-DOPE-P5-MPL) induced an effective cellular immune response characterizedwith the higher IFN-γ producing CD8+ cells and CTL activity As

Fig 4 Antigen-specific CTL response induced by various formulations at two dif-ferent ratios of effector to target cells (ET) was assessed using an in vitro CTL activityassay Splenocytes isolated from mice (four in each group) were incubated with CalceinAM-loaded rHER2neu-expressing TUBO tumor cells and rHER2neu-expressing neg-ative CT26 cells (see method)The data indicate the mean plusmn SEM (n = 4) denotessignificant difference from all other formulations at both various effector-to-targetratios

A B

Fig 5 Protective effects of vaccination with different formulations in BALBc mice against a TUBO tumor model (A) Immunized mice (six in each group) were challenged14 days post last booster with 5 times 105 TUBO cells Tumor size was calculated based on three dimensions The values are means of tumor size plusmn SEM (n = 6) (B) Effects ofimmunization on survival time were monitored for a period of 84 days among BALBc mice (n = 6) denotes significant difference from all other formulations

Table 3Therapeutic efficacy data of different liposomal vaccine formulations in TUBO tumormice model (n = 6)

Formulation MSTa (day) TTEb (day plusmn SD) TGDc

Buffer 525 4797 plusmn 881 ndashP5 56 5567 plusmn 1344 1858Lip-DOPE-MPL 63 5803 plusmn 836 3190Lip-P5 665 6267 plusmn 1254 3531Lip-P5-MPL 595 5703 plusmn 1318 2357Lip-DOPE-P5 70 6910 plusmn 1190 5416Lip-DOPE-P5-MPL Indefinable 17545 plusmn 3116 8041

Denotes significant difference from all other formulationsa Median survival timeb Time to reach end pointc Tumor growth delay

58 S Shariat et alCancer Letters 355 (2014) 54ndash60

results have shown the pair of MPL and DOPE used in liposomalformulation had synergic effect on promoting vaccine efficacy whileusing one of them individually in the lipid carrier (Lip-P5-MPL orLip-DOPE-P5 formulations) was impotent to induce immune re-sponse Due to the lipidic structure of MPL and DOPE whichfacilitated incorporation to liposomes and the safe use of anionicliposomes containing MPL in humans [13] Lip-DOPE-P5-MPL for-mulation might be superior to vaccine delivery systems such ascationic liposomes containing CpG ODN or LPDs employed for in-duction of CTL response against HER2-derived peptide antigens[1638]

The success of Lip-DOPE-P5-MPL formulation to elicit a robustCTL response against P5 peptide containing just MHC class I epitopesof HER2neu without the use of T-helper epitope can be accordantwith the claim that MPL has the stimulatory action on APCs [1035]Although formulations containing both CTL and T helper epitopesderived from Her2neu antigen in combination with GM-CSF couldinduce effective CTL responses [3940] and confirmed the claim thatCD4 T cell help was required to activate CTLs [4142] our findingsdemonstrated that this requirement might be dependent on howpeptide vaccines were formulated MPL has been shown to becapable of provoking DCs to produce co-stimulatory molecules andsecrete inflammatory cytokines such as IL-6 IFN-γ and IL-12 whichare essential for activation of T cells [103543ndash45] CTL inductiondue to the stimulatory action of TLR agonists on APCs was previ-ously observed about CpG ODN adjuvant [38]

In conclusion our results show that Lip-DOPE-P5-MPL is an ef-fective formulation for the preparation of a peptide-based HER2neu vaccine Liposomes composed of DMPCDMPGCholDOPEcontaining MPL can deliver peptide antigen to the cytosol and induceCTL response that can be used prophylactically to reduce tumorgrowth The benefits of this formulation (easy manufacturing processand safe use in human) can make it a potential candidate to alter-native ones for developing liposomal vaccines in terms of antitumortherapies in breast cancers in which HER2neu antigen overexpresses

Conflict of interest

The authors declare no conflict of interest

Acknowledgements

We are grateful for the financial support from Shahid BeheshtiUniversity of Medical Sciences Biotechnology Research Center andNanotechnology Research Center Mashhad University of MedicalSciences We would also like to thank Azam Abbasi and Zahra Saberifor their excellent technical assistance This study was a part of SShariatrsquos PhD dissertation

References

[1] J Couzin-Frankel Breakthrough of the year 2013 Cancer immunotherapyScience 342 (2013) 1432ndash1433

[2] HL Hanson DL Donermeyer H Ikeda JM White V Shankaran LJ Old et alEradication of established tumors by CD8+ T cell adoptive immunotherapyImmunity 13 (2000) 265ndash276

[3] K Matsui LA OrsquoMara PM Allen Successful elimination of large establishedtumors and avoidance of antigen-loss variants by aggressive adoptive T cellimmunotherapy Int Immunol 15 (2003) 797ndash805

[4] RM Steinman Decisions about dendritic cells past present and future AnnuRev Immunol 30 (2012) 1ndash22

[5] K Palucka J Banchereau Human dendritic cell subsets in vaccination CurrOpin Immunol 25 (2013) 396ndash402

[6] J Parkin B Cohen An overview of the immune system Lancet 357 (2001)1777ndash1789

[7] DS Watson AN Endsley L Huang Design considerations for liposomalvaccines influence of formulation parameters on antibody and cell-mediatedimmune responses to liposome associated antigens Vaccine 30 (2012) 2256ndash2272

[8] CR Alving Liposomes as carriers of antigens and adjuvants J ImmunolMethods 140 (1991) 1ndash13

[9] P Nordly HB Madsen HM Nielsen C Foged Status and future prospects oflipid-based particulate delivery systems as vaccine adjuvants and theircombination with immunostimulators Expert Opin Drug Deliv 6 (2009)657ndash672

[10] JT Ulrich KR Myers Monophosphoryl lipid A as an adjuvant Past experiencesand new directions Pharm Biotechnol 6 (1995) 495ndash524

[11] CR Alving Lipopolysaccharide lipid A and liposomes containing lipid A asimmunologic adjuvants Immunobiology 187 (1993) 430ndash446

[12] CR Casella TC Mitchell Putting endotoxin to work for us monophosphoryllipid A as a safe and effective vaccine adjuvant Cell Mol Life Sci 65 (2008)3231ndash3240

[13] E Vacchelli L Galluzzi A Eggermont WH Fridman J Galon C Sautes-Fridmanet al Trial watch FDA-approved Toll-like receptor agonists for cancer therapyOncoimmunology 1 (2012) 894ndash907

[14] Y Yarden Biology of HER2 and its importance in breast cancer Oncology 61(Suppl 2) (2001) 1ndash13

[15] CN Baxevanis NN Sotiriadou AD Gritzapis PA Sotiropoulou SA Perez NTCacoullos et al Immunogenic HER-2neu peptides as tumor vaccines CancerImmunol Immunother 55 (2006) 85ndash95

[16] SA Jalali M Sankian J Tavakkol-Afshari MR Jaafari Induction of tumor-specific immunity by multi-epitope rat HER2neu-derived peptides encapsulatedin LPD nanoparticles Nanomedicine 8 (2012) 692ndash701

[17] AA Levin A review of the issues in the pharmacokinetics and toxicology ofphosphorothioate antisense oligonucleotides Biochim Biophys Acta 1489(1999) 69ndash84

[18] S Shariat A Badiee MR Jaafari SA Mortazavi Optimization of a method toprepare liposomes containing HER2neu- derived peptide as a vaccine deliverysystem for breast cancer Iran J Pharm Res 13 (2014) 15ndash25

[19] P Harmon D Cabral-Lilly RA Reed FP Maurio JC Franklin A Janoff Therelease and detection of endotoxin from liposomes Anal Biochem 250 (1997)139ndash146

[20] GR Bartlett Phosphorus assay in column chromatography J Biol Chem 234(1959) 466ndash468

[21] R Lichtenfels WE Biddison H Schulz AB Vogt R Martin CARE-LASS(calcein-release-assay) an improved fluorescence-based test system tomeasure cytotoxic T lymphocyte activity J Immunol Methods 172 (1994)227ndash239

[22] Z Huang MR Jaafari FC Szoka Jr Disterolphospholipids nonexchangeablelipids and their application to liposomal drug delivery Angew Chem Int EdEngl 48 (2009) 4146ndash4149

[23] T Schluep J Hwang J Cheng JD Heidel DW Bartlett B Hollister et alPreclinical efficacy of the camptothecin-polymer conjugate IT-101 in multiplecancer models Clin Cancer Res 12 (2006) 1606ndash1614

[24] MF Bachmann GT Jennings Vaccine delivery a matter of size geometrykinetics and molecular patterns Nat Rev Immunol 10 (2010) 787ndash796

[25] AH Hale H-2 antigens incorporated into phospholipid vesicles elicit specificallogeneic cytotoxic T lymphocytes Cell Immunol 55 (1980) 328ndash341

[26] L Raphael BH Tom Liposome facilitated xenogeneic approach for studyinghuman colon cancer immunity carrier and adjuvant effect of liposomes ClinExp Immunol 55 (1984) 1ndash13

[27] RM Steinman L Bonifaz S Fujii K Liu D Bonnyay S Yamazaki et al Theinnate functions of dendritic cells in peripheral lymphoid tissues Adv Exp MedBiol 560 (2005) 83ndash97

[28] R Reddy F Zhou L Huang F Carbone M Bevan BT Rouse pH sensitiveliposomes provide an efficient means of sensitizing target cells to class Irestricted CTL recognition of a soluble protein J Immunol Methods 141 (1991)157ndash163

[29] S Nair F Zhou R Reddy L Huang BT Rouse Soluble proteins delivered todendritic cells via pH-sensitive liposomes induce primary cytotoxic Tlymphocyte responses in vitro J Exp Med 175 (1992) 609ndash612

[30] JS Chang MJ Choi HS Cheong K Kim Development of Th1-mediated CD8+effector T cells by vaccination with epitope peptides encapsulated in pH-sensitive liposomes Vaccine 19 (2001) 3608ndash3614

[31] L Luo Y Li JS Chang SY Cho TY Kim MJ Choi et al Induction of V3-specificcytotoxic T lymphocyte responses by HIV gag particles carrying multipleimmunodominant V3 epitopes of gp120 Virology 240 (1998) 316ndash325

[32] DP Siegel RM Epand The mechanism of lamellar-to-inverted hexagonal phasetransitions in phosphatidylethanolamine implications for membrane fusionmechanisms Biophys J 73 (1997) 3089ndash3111

[33] J Connor MB Yatvin L Huang pH-sensitive liposomes acid-induced liposomefusion Proc Natl Acad Sci USA 81 (1984) 1715ndash1718

[34] N Duumlzguumlnes RM Straubinger PA Baldwin DS Friend D PapahadjopoulosProton-induced fusion of oleic acid-phosphatidylethanolamine liposomesBiochemistry 24 (1985) 3091ndash3098

[35] DJ Marciani Vaccine adjuvants role and mechanisms of action in vaccineimmunogenicity Drug Discov Today 8 (2003) 934ndash943

[36] B Guy The perfect mix recent progress in adjuvant research Nat RevMicrobiol 5 (2007) 505ndash517

[37] VE Schijns EC Lavelle Trends in vaccine adjuvants Expert Rev Vaccines 10(2011) 539ndash550

[38] WM Li WH Dragowska MB Bally MP Schutze-Redelmeier Effectiveinduction of CD8+ T-cell response using CpG oligodeoxynucleotides andHER-2neu-derived peptide co-encapsulated in liposomes Vaccine 21 (2003)3319ndash3329

59S Shariat et alCancer Letters 355 (2014) 54ndash60

containing 2 Triton X-100 were added to the wells to determine the minimum andmaximum release by target cells respectively Fluorescence in supernatants was readon a fluorimeter (FLx800 BioTek Instruments Inc USA) with excitation at 485 nmand emission at 538 nm The specific lysis was calculated as follows percentage ofspecific lysis = (release by CTLs minus minimum release by targets)(maximum releaseby targets minus minimum release by targets) CT26 cells which labeled similarly to theTUBO cells were used as negative control to prove that cytotoxic activity isspecific

In vivo tumor protection assay

The immunized mice (six per group) were challenged on day 14 post last vac-cination via subcutaneous injection in the right flank with 5 times 105 TUBO cells in 50 μlPBS buffer The tumor volume ([length times width times height] times 05) the time to reach endpoint (TTE) (from the equation of the line obtained by exponential regression of thetumor growth curve) and the percent of tumor growth delay (TGD) (based on thedifference between the median TTE of treatment group (T) and the median TTE ofthe control group (C) (TGD = [(T minus C)C] times 100]) were calculated for each mouse[2223] For ethical consideration mice were sacrificed when (a) the tumor volumewas greater than 1000 mm3 (b) the body weight loss was over 15 of initial weightor (c) the mice became lethargic or sick and unable to feed

Statistical analysis

The results were analyzed by one-way ANOVA and Tukey test to assess the sig-nificance of the differences among various formulations Mouse survival was analyzedby log-rank test (GraphPad Prism version 5 San Diego California) Results with P lt 005were considered to be statistically significant

Results

Synthesis of P5-PEG-DSPE

Formation of P5-PEG-DSPE was identified by thin layer chro-matography Disappearance of PEG-DSPE spot from the reactionmixture was confirmed by iodine vapor (Fig 1A) The extent ofunconjugated (free) peptide was determined using HPLC post re-action with PEG-DSPE and the results indicated almost completereaction between maleamide group in PEG-DSPE and thiol on P5(Fig 1B)

Physical properties of liposomal formulations

P5-PEG-DSPE was incorporated into different liposomal formu-lations to induce an effective CTL response For each formulationphysical property including liposome size polydispersity index (pdI)and zeta potential was determined as shown in Table 1

The particle size of all liposomal formulations ranged from 110to 150 nm in diameter which was desirable for vaccine formula-tions [24] Negatively charged liposomes were also homogenous andhad a uniform size with monomodal distribution (pdI lt 02)

Content of P5 peptide and MPL in liposomal formulations

Since the dose of peptide antigen and adjuvant can signifi-cantly influence the efficacy of formulations P5 peptide andMPL content were accurately determined in different liposomal for-mulations (Table 2) Based on the lipid dose of 5 μmol a similarpeptide and MPL doses per mouse were administered for eachformulation

Fig 1 Confirmation of P5 peptide conjugation to Maleimide-PEG-DSPE using TLC (A) and HPLC (B) (A) A TLC plate was placed in TLC chamber containing mobile phasecomposed of chloroform methanol and water at 90182 (vv) Disappearance of PEG-DSPE spot from the reaction mixture was confirmed by iodine vapor (B I) Standardfree peptide eluted with a retention time of ~12 minutes (B II) The extent of unconjugated (free) peptide was determined post reaction with PEG-DSPE (see method)

Table 1Vesicle size pdI and zeta potential of liposomal formulations (n = 3 mean plusmn SD)

Formulation Vesicle size(nm)

pdI Zeta potential(mV)

Lip-DOPE-MPL (DMPCDMPGCholDOPEMPL)

1263 plusmn 35 0138 plusmn 0019 minus446 plusmn 128

Lip-P5 (DMPCDMPGCholP5) 1357 plusmn 61 0173 plusmn 0014 minus417 plusmn 165Lip-P5-MPL (DMPCDMPG

CholP5MPL)1289 plusmn 63 0147 plusmn 0011 minus474 plusmn 146

Lip-DOPE-P5 (DMPCDMPGCholDOPEP5)

1423 plusmn 56 0184 plusmn 0008 minus424 plusmn 221

Lip-DOPE-P5-MPL (DMPCDMPGCholDOPEP5MPL)

1327 plusmn 93 0176 plusmn 0026 minus447 plusmn 255

56 S Shariat et alCancer Letters 355 (2014) 54ndash60

Induction of IFN-γ response by Lip-DOPE-P5-MPL formulation

The results showed that splenocytes isolated from the mice im-munized with liposomes composed of DMPCDMPGCholDOPEcontaining P5 peptide and MPL (Lip-DOPE-P5-MPL) secreted a sig-nificantly higher amount of IFN-γ than the other liposomalformulations P5 peptide alone and HEPES buffer (Fig 2A) None ofthe liposomal formulations induced considerable IL-4 response inmice (Fig 2B)

Induction of CD8+ response by Lip-DOPE-P5-MPL formulation

Flow cytometric analysis using CD8 CD4 and IFN-γ markers dem-onstrated CD8+ T lymphocytes mostly contributed to IFN-γproduction (Fig 3A and B) Moreover the results showed Lip-DOPE-P5-MPL formulation induced a significantly higher level productionof IFN-γ in CD8+ lymphocytes (higher MFI level) which repre-sented a higher number of IFN-γ producing cells in CD8+ populationor higher CTL population in comparison with other groups (Fig 3A)Flow cytometric results also showed IL-4 production in CD+4 cellsthat implies T cell-dependent humoral immunity was not inducedsignificantly in all groups (Fig 3C)

Induction of antigen-specific CTL response byLip-DOPE-P5-MPL formulation

In vitro CTL activity assay using rHER2neu-expressingTUBO tumor cells indicated immunization with Lip-DOPE-P5-MPLformulation generated significantly higher CTL response to P5antigen than other formulations (Fig 4) The CTL activity was es-tablished significantly at both various effector-to-target ratios The

Table 2Content of P5 peptide and MPL in liposomal formulations

Formulation P5 content(μgμmollipid)

MPL content(μgμmollipid)

P5 dose(μg permouse)

MPL dose(μg permouse)

Lip-DOPE-MPL ndash 491 ndash 2455Lip-P5 185 ndash 925 ndashLip-P5-MPL 189 482 945 2410Lip-DOPE-P5 196 ndash 980 ndashLip-DOPE-P5-MPL 192 508 960 2540

P5 peptide dose and MPL dose were determined based on a lipid dose of 5 μmolgiven per mouse

Fig 2 The efficacy of different liposomal formulations in inducing IFN-γ (A) and IL-4 (B) production BALBc mice (10 per group) were immunized three times at two-weekintervals with different liposomal formulations P5 peptide alone or HEPES buffer On day 14 post last booster four mice from each group were sacrificed and their splenocyteswere restimulated with P5 peptide IFN-γ and IL-4 release from splenocytes induced by different liposomal formulations was determined using ELISpot assay The data in-dicate the mean plusmn SEM (n = 4) denotes significant difference from all other formulations

Fig 3 Geometric mean fluorescence intensity (MFI) level for IFN-γ in gated CD8 (A) and CD4 (B) lymphocyte populations and MFI level for IL-4 in gated CD4s (C) Isolatedsplenocytes of immunized mice were re-stimulated in vitro with PMAionomycin and stained with CD4 CD8 IFN-γ and IL-4 markers MFI level for IFN-γ and IL-4 in gatedpopulations were determined by flow cytometric analysis The data indicate the mean plusmn SEM (n = 4) and denote significant difference from buffer and all other for-mulations respectively

57S Shariat et alCancer Letters 355 (2014) 54ndash60

cytotoxicity activity induced by Lip-DOPE-P5-MPL was specificagainst rHER2neu CTL response was not observed against rHER2neu-expressing negative CT26 cells (Fig 4)

Anti tumor effects of Lip-DOPE-P5-MPL vaccination in BALBc

Lip-DOPE-P5-MPL formulation had a superior tumor growth in-hibition in the TUBO tumor mice model (Fig 5A) The survival timewas also significantly prolonged in mice following Lip-DOPE-P5-MPL vaccination compared to the other formulations (Fig 5B) Sincenone of the mice vaccinated with Lip-DOPE-P5-MPL died during theexperiment median survival was indefinable in this group Mediansurvival time (MST) as well as TTE and TGD for each treatmentgroup are summarized in Table 3

Discussion

In the present study we attempted to enhance immunogenic-ity and adjuvanicity of P5 peptide a synthetic peptide containingCTL multi-epitope from rHER2neu protein by conjugating with

different liposomal formulations The results showed P5 peptide con-jugating with liposomes alone (Lip-P5) was incapable of inducingany immune response Although some studies reported liposomesalone could elicit CTL response against encapsulated peptide anti-gens [2526] this formulation could have been used exclusively asa carrier for antigen delivery with no potential to conduct anti-gens into the cytosol Lip-P5 formulation had a particle size of 110ndash150 nm that is needed for efficiently draining to lymph nodes whereCD8+ lymphoid DCs are present [2427]

Inclusion of a pH sensitive lipid like DOPE in the liposome struc-ture has been frequently demonstrated as an efficient strategy tointroduce antigens into MHC class I pathway [28ndash31] After endo-cytosis at the pH of the endosomal compartment the transition ofDOPE from lamellar to hexagonal phase occurs It induces the fusionof liposomes with the endosomal membrane and subsequently li-posomal antigens are released toward the cytosol [32ndash34] Howeveras results have shown inclusion of DOPE alone in liposomal for-mulation (Lip-DOPE-P5) was not sufficient to elicit an effectiveimmune response Lip-DOPE-P5 formulation might have been ableto deliver P5 antigen to MHC class I molecules successfully howeveronce the peptide antigen was presented to CD8+ lymphocytes byAPCs presence of co-stimulatory molecules on the APCs was alsorequired for activation of CD8 cells to produce CTLs [35ndash37] MPLcan induce intracellular signaling pathways leading to productionof these co-stimulatory molecules through TLR4 stimulation [35]Co-formulation of MPL and DOPE in liposomes (Lip-DOPE-P5-MPL) induced an effective cellular immune response characterizedwith the higher IFN-γ producing CD8+ cells and CTL activity As

Fig 4 Antigen-specific CTL response induced by various formulations at two dif-ferent ratios of effector to target cells (ET) was assessed using an in vitro CTL activityassay Splenocytes isolated from mice (four in each group) were incubated with CalceinAM-loaded rHER2neu-expressing TUBO tumor cells and rHER2neu-expressing neg-ative CT26 cells (see method)The data indicate the mean plusmn SEM (n = 4) denotessignificant difference from all other formulations at both various effector-to-targetratios

A B

Fig 5 Protective effects of vaccination with different formulations in BALBc mice against a TUBO tumor model (A) Immunized mice (six in each group) were challenged14 days post last booster with 5 times 105 TUBO cells Tumor size was calculated based on three dimensions The values are means of tumor size plusmn SEM (n = 6) (B) Effects ofimmunization on survival time were monitored for a period of 84 days among BALBc mice (n = 6) denotes significant difference from all other formulations

Table 3Therapeutic efficacy data of different liposomal vaccine formulations in TUBO tumormice model (n = 6)

Formulation MSTa (day) TTEb (day plusmn SD) TGDc

Buffer 525 4797 plusmn 881 ndashP5 56 5567 plusmn 1344 1858Lip-DOPE-MPL 63 5803 plusmn 836 3190Lip-P5 665 6267 plusmn 1254 3531Lip-P5-MPL 595 5703 plusmn 1318 2357Lip-DOPE-P5 70 6910 plusmn 1190 5416Lip-DOPE-P5-MPL Indefinable 17545 plusmn 3116 8041

Denotes significant difference from all other formulationsa Median survival timeb Time to reach end pointc Tumor growth delay

58 S Shariat et alCancer Letters 355 (2014) 54ndash60

results have shown the pair of MPL and DOPE used in liposomalformulation had synergic effect on promoting vaccine efficacy whileusing one of them individually in the lipid carrier (Lip-P5-MPL orLip-DOPE-P5 formulations) was impotent to induce immune re-sponse Due to the lipidic structure of MPL and DOPE whichfacilitated incorporation to liposomes and the safe use of anionicliposomes containing MPL in humans [13] Lip-DOPE-P5-MPL for-mulation might be superior to vaccine delivery systems such ascationic liposomes containing CpG ODN or LPDs employed for in-duction of CTL response against HER2-derived peptide antigens[1638]

The success of Lip-DOPE-P5-MPL formulation to elicit a robustCTL response against P5 peptide containing just MHC class I epitopesof HER2neu without the use of T-helper epitope can be accordantwith the claim that MPL has the stimulatory action on APCs [1035]Although formulations containing both CTL and T helper epitopesderived from Her2neu antigen in combination with GM-CSF couldinduce effective CTL responses [3940] and confirmed the claim thatCD4 T cell help was required to activate CTLs [4142] our findingsdemonstrated that this requirement might be dependent on howpeptide vaccines were formulated MPL has been shown to becapable of provoking DCs to produce co-stimulatory molecules andsecrete inflammatory cytokines such as IL-6 IFN-γ and IL-12 whichare essential for activation of T cells [103543ndash45] CTL inductiondue to the stimulatory action of TLR agonists on APCs was previ-ously observed about CpG ODN adjuvant [38]

In conclusion our results show that Lip-DOPE-P5-MPL is an ef-fective formulation for the preparation of a peptide-based HER2neu vaccine Liposomes composed of DMPCDMPGCholDOPEcontaining MPL can deliver peptide antigen to the cytosol and induceCTL response that can be used prophylactically to reduce tumorgrowth The benefits of this formulation (easy manufacturing processand safe use in human) can make it a potential candidate to alter-native ones for developing liposomal vaccines in terms of antitumortherapies in breast cancers in which HER2neu antigen overexpresses

Conflict of interest

The authors declare no conflict of interest

Acknowledgements

We are grateful for the financial support from Shahid BeheshtiUniversity of Medical Sciences Biotechnology Research Center andNanotechnology Research Center Mashhad University of MedicalSciences We would also like to thank Azam Abbasi and Zahra Saberifor their excellent technical assistance This study was a part of SShariatrsquos PhD dissertation

References

[1] J Couzin-Frankel Breakthrough of the year 2013 Cancer immunotherapyScience 342 (2013) 1432ndash1433

[2] HL Hanson DL Donermeyer H Ikeda JM White V Shankaran LJ Old et alEradication of established tumors by CD8+ T cell adoptive immunotherapyImmunity 13 (2000) 265ndash276

[3] K Matsui LA OrsquoMara PM Allen Successful elimination of large establishedtumors and avoidance of antigen-loss variants by aggressive adoptive T cellimmunotherapy Int Immunol 15 (2003) 797ndash805

[4] RM Steinman Decisions about dendritic cells past present and future AnnuRev Immunol 30 (2012) 1ndash22

[5] K Palucka J Banchereau Human dendritic cell subsets in vaccination CurrOpin Immunol 25 (2013) 396ndash402

[6] J Parkin B Cohen An overview of the immune system Lancet 357 (2001)1777ndash1789

[7] DS Watson AN Endsley L Huang Design considerations for liposomalvaccines influence of formulation parameters on antibody and cell-mediatedimmune responses to liposome associated antigens Vaccine 30 (2012) 2256ndash2272

[8] CR Alving Liposomes as carriers of antigens and adjuvants J ImmunolMethods 140 (1991) 1ndash13

[9] P Nordly HB Madsen HM Nielsen C Foged Status and future prospects oflipid-based particulate delivery systems as vaccine adjuvants and theircombination with immunostimulators Expert Opin Drug Deliv 6 (2009)657ndash672

[10] JT Ulrich KR Myers Monophosphoryl lipid A as an adjuvant Past experiencesand new directions Pharm Biotechnol 6 (1995) 495ndash524

[11] CR Alving Lipopolysaccharide lipid A and liposomes containing lipid A asimmunologic adjuvants Immunobiology 187 (1993) 430ndash446

[12] CR Casella TC Mitchell Putting endotoxin to work for us monophosphoryllipid A as a safe and effective vaccine adjuvant Cell Mol Life Sci 65 (2008)3231ndash3240

[13] E Vacchelli L Galluzzi A Eggermont WH Fridman J Galon C Sautes-Fridmanet al Trial watch FDA-approved Toll-like receptor agonists for cancer therapyOncoimmunology 1 (2012) 894ndash907

[14] Y Yarden Biology of HER2 and its importance in breast cancer Oncology 61(Suppl 2) (2001) 1ndash13

[15] CN Baxevanis NN Sotiriadou AD Gritzapis PA Sotiropoulou SA Perez NTCacoullos et al Immunogenic HER-2neu peptides as tumor vaccines CancerImmunol Immunother 55 (2006) 85ndash95

[16] SA Jalali M Sankian J Tavakkol-Afshari MR Jaafari Induction of tumor-specific immunity by multi-epitope rat HER2neu-derived peptides encapsulatedin LPD nanoparticles Nanomedicine 8 (2012) 692ndash701

[17] AA Levin A review of the issues in the pharmacokinetics and toxicology ofphosphorothioate antisense oligonucleotides Biochim Biophys Acta 1489(1999) 69ndash84

[18] S Shariat A Badiee MR Jaafari SA Mortazavi Optimization of a method toprepare liposomes containing HER2neu- derived peptide as a vaccine deliverysystem for breast cancer Iran J Pharm Res 13 (2014) 15ndash25

[19] P Harmon D Cabral-Lilly RA Reed FP Maurio JC Franklin A Janoff Therelease and detection of endotoxin from liposomes Anal Biochem 250 (1997)139ndash146

[20] GR Bartlett Phosphorus assay in column chromatography J Biol Chem 234(1959) 466ndash468

[21] R Lichtenfels WE Biddison H Schulz AB Vogt R Martin CARE-LASS(calcein-release-assay) an improved fluorescence-based test system tomeasure cytotoxic T lymphocyte activity J Immunol Methods 172 (1994)227ndash239

[22] Z Huang MR Jaafari FC Szoka Jr Disterolphospholipids nonexchangeablelipids and their application to liposomal drug delivery Angew Chem Int EdEngl 48 (2009) 4146ndash4149

[23] T Schluep J Hwang J Cheng JD Heidel DW Bartlett B Hollister et alPreclinical efficacy of the camptothecin-polymer conjugate IT-101 in multiplecancer models Clin Cancer Res 12 (2006) 1606ndash1614

[24] MF Bachmann GT Jennings Vaccine delivery a matter of size geometrykinetics and molecular patterns Nat Rev Immunol 10 (2010) 787ndash796

[25] AH Hale H-2 antigens incorporated into phospholipid vesicles elicit specificallogeneic cytotoxic T lymphocytes Cell Immunol 55 (1980) 328ndash341

[26] L Raphael BH Tom Liposome facilitated xenogeneic approach for studyinghuman colon cancer immunity carrier and adjuvant effect of liposomes ClinExp Immunol 55 (1984) 1ndash13

[27] RM Steinman L Bonifaz S Fujii K Liu D Bonnyay S Yamazaki et al Theinnate functions of dendritic cells in peripheral lymphoid tissues Adv Exp MedBiol 560 (2005) 83ndash97

[28] R Reddy F Zhou L Huang F Carbone M Bevan BT Rouse pH sensitiveliposomes provide an efficient means of sensitizing target cells to class Irestricted CTL recognition of a soluble protein J Immunol Methods 141 (1991)157ndash163

[29] S Nair F Zhou R Reddy L Huang BT Rouse Soluble proteins delivered todendritic cells via pH-sensitive liposomes induce primary cytotoxic Tlymphocyte responses in vitro J Exp Med 175 (1992) 609ndash612

[30] JS Chang MJ Choi HS Cheong K Kim Development of Th1-mediated CD8+effector T cells by vaccination with epitope peptides encapsulated in pH-sensitive liposomes Vaccine 19 (2001) 3608ndash3614

[31] L Luo Y Li JS Chang SY Cho TY Kim MJ Choi et al Induction of V3-specificcytotoxic T lymphocyte responses by HIV gag particles carrying multipleimmunodominant V3 epitopes of gp120 Virology 240 (1998) 316ndash325

[32] DP Siegel RM Epand The mechanism of lamellar-to-inverted hexagonal phasetransitions in phosphatidylethanolamine implications for membrane fusionmechanisms Biophys J 73 (1997) 3089ndash3111

[33] J Connor MB Yatvin L Huang pH-sensitive liposomes acid-induced liposomefusion Proc Natl Acad Sci USA 81 (1984) 1715ndash1718

[34] N Duumlzguumlnes RM Straubinger PA Baldwin DS Friend D PapahadjopoulosProton-induced fusion of oleic acid-phosphatidylethanolamine liposomesBiochemistry 24 (1985) 3091ndash3098

[35] DJ Marciani Vaccine adjuvants role and mechanisms of action in vaccineimmunogenicity Drug Discov Today 8 (2003) 934ndash943

[36] B Guy The perfect mix recent progress in adjuvant research Nat RevMicrobiol 5 (2007) 505ndash517

[37] VE Schijns EC Lavelle Trends in vaccine adjuvants Expert Rev Vaccines 10(2011) 539ndash550

[38] WM Li WH Dragowska MB Bally MP Schutze-Redelmeier Effectiveinduction of CD8+ T-cell response using CpG oligodeoxynucleotides andHER-2neu-derived peptide co-encapsulated in liposomes Vaccine 21 (2003)3319ndash3329

59S Shariat et alCancer Letters 355 (2014) 54ndash60

Induction of IFN-γ response by Lip-DOPE-P5-MPL formulation

The results showed that splenocytes isolated from the mice im-munized with liposomes composed of DMPCDMPGCholDOPEcontaining P5 peptide and MPL (Lip-DOPE-P5-MPL) secreted a sig-nificantly higher amount of IFN-γ than the other liposomalformulations P5 peptide alone and HEPES buffer (Fig 2A) None ofthe liposomal formulations induced considerable IL-4 response inmice (Fig 2B)

Induction of CD8+ response by Lip-DOPE-P5-MPL formulation

Flow cytometric analysis using CD8 CD4 and IFN-γ markers dem-onstrated CD8+ T lymphocytes mostly contributed to IFN-γproduction (Fig 3A and B) Moreover the results showed Lip-DOPE-P5-MPL formulation induced a significantly higher level productionof IFN-γ in CD8+ lymphocytes (higher MFI level) which repre-sented a higher number of IFN-γ producing cells in CD8+ populationor higher CTL population in comparison with other groups (Fig 3A)Flow cytometric results also showed IL-4 production in CD+4 cellsthat implies T cell-dependent humoral immunity was not inducedsignificantly in all groups (Fig 3C)

Induction of antigen-specific CTL response byLip-DOPE-P5-MPL formulation

In vitro CTL activity assay using rHER2neu-expressingTUBO tumor cells indicated immunization with Lip-DOPE-P5-MPLformulation generated significantly higher CTL response to P5antigen than other formulations (Fig 4) The CTL activity was es-tablished significantly at both various effector-to-target ratios The

Table 2Content of P5 peptide and MPL in liposomal formulations

Formulation P5 content(μgμmollipid)

MPL content(μgμmollipid)

P5 dose(μg permouse)

MPL dose(μg permouse)

Lip-DOPE-MPL ndash 491 ndash 2455Lip-P5 185 ndash 925 ndashLip-P5-MPL 189 482 945 2410Lip-DOPE-P5 196 ndash 980 ndashLip-DOPE-P5-MPL 192 508 960 2540

P5 peptide dose and MPL dose were determined based on a lipid dose of 5 μmolgiven per mouse

Fig 2 The efficacy of different liposomal formulations in inducing IFN-γ (A) and IL-4 (B) production BALBc mice (10 per group) were immunized three times at two-weekintervals with different liposomal formulations P5 peptide alone or HEPES buffer On day 14 post last booster four mice from each group were sacrificed and their splenocyteswere restimulated with P5 peptide IFN-γ and IL-4 release from splenocytes induced by different liposomal formulations was determined using ELISpot assay The data in-dicate the mean plusmn SEM (n = 4) denotes significant difference from all other formulations

Fig 3 Geometric mean fluorescence intensity (MFI) level for IFN-γ in gated CD8 (A) and CD4 (B) lymphocyte populations and MFI level for IL-4 in gated CD4s (C) Isolatedsplenocytes of immunized mice were re-stimulated in vitro with PMAionomycin and stained with CD4 CD8 IFN-γ and IL-4 markers MFI level for IFN-γ and IL-4 in gatedpopulations were determined by flow cytometric analysis The data indicate the mean plusmn SEM (n = 4) and denote significant difference from buffer and all other for-mulations respectively

57S Shariat et alCancer Letters 355 (2014) 54ndash60

cytotoxicity activity induced by Lip-DOPE-P5-MPL was specificagainst rHER2neu CTL response was not observed against rHER2neu-expressing negative CT26 cells (Fig 4)

Anti tumor effects of Lip-DOPE-P5-MPL vaccination in BALBc

Lip-DOPE-P5-MPL formulation had a superior tumor growth in-hibition in the TUBO tumor mice model (Fig 5A) The survival timewas also significantly prolonged in mice following Lip-DOPE-P5-MPL vaccination compared to the other formulations (Fig 5B) Sincenone of the mice vaccinated with Lip-DOPE-P5-MPL died during theexperiment median survival was indefinable in this group Mediansurvival time (MST) as well as TTE and TGD for each treatmentgroup are summarized in Table 3

Discussion

In the present study we attempted to enhance immunogenic-ity and adjuvanicity of P5 peptide a synthetic peptide containingCTL multi-epitope from rHER2neu protein by conjugating with

different liposomal formulations The results showed P5 peptide con-jugating with liposomes alone (Lip-P5) was incapable of inducingany immune response Although some studies reported liposomesalone could elicit CTL response against encapsulated peptide anti-gens [2526] this formulation could have been used exclusively asa carrier for antigen delivery with no potential to conduct anti-gens into the cytosol Lip-P5 formulation had a particle size of 110ndash150 nm that is needed for efficiently draining to lymph nodes whereCD8+ lymphoid DCs are present [2427]

Inclusion of a pH sensitive lipid like DOPE in the liposome struc-ture has been frequently demonstrated as an efficient strategy tointroduce antigens into MHC class I pathway [28ndash31] After endo-cytosis at the pH of the endosomal compartment the transition ofDOPE from lamellar to hexagonal phase occurs It induces the fusionof liposomes with the endosomal membrane and subsequently li-posomal antigens are released toward the cytosol [32ndash34] Howeveras results have shown inclusion of DOPE alone in liposomal for-mulation (Lip-DOPE-P5) was not sufficient to elicit an effectiveimmune response Lip-DOPE-P5 formulation might have been ableto deliver P5 antigen to MHC class I molecules successfully howeveronce the peptide antigen was presented to CD8+ lymphocytes byAPCs presence of co-stimulatory molecules on the APCs was alsorequired for activation of CD8 cells to produce CTLs [35ndash37] MPLcan induce intracellular signaling pathways leading to productionof these co-stimulatory molecules through TLR4 stimulation [35]Co-formulation of MPL and DOPE in liposomes (Lip-DOPE-P5-MPL) induced an effective cellular immune response characterizedwith the higher IFN-γ producing CD8+ cells and CTL activity As

Fig 4 Antigen-specific CTL response induced by various formulations at two dif-ferent ratios of effector to target cells (ET) was assessed using an in vitro CTL activityassay Splenocytes isolated from mice (four in each group) were incubated with CalceinAM-loaded rHER2neu-expressing TUBO tumor cells and rHER2neu-expressing neg-ative CT26 cells (see method)The data indicate the mean plusmn SEM (n = 4) denotessignificant difference from all other formulations at both various effector-to-targetratios

A B

Fig 5 Protective effects of vaccination with different formulations in BALBc mice against a TUBO tumor model (A) Immunized mice (six in each group) were challenged14 days post last booster with 5 times 105 TUBO cells Tumor size was calculated based on three dimensions The values are means of tumor size plusmn SEM (n = 6) (B) Effects ofimmunization on survival time were monitored for a period of 84 days among BALBc mice (n = 6) denotes significant difference from all other formulations

Table 3Therapeutic efficacy data of different liposomal vaccine formulations in TUBO tumormice model (n = 6)

Formulation MSTa (day) TTEb (day plusmn SD) TGDc

Buffer 525 4797 plusmn 881 ndashP5 56 5567 plusmn 1344 1858Lip-DOPE-MPL 63 5803 plusmn 836 3190Lip-P5 665 6267 plusmn 1254 3531Lip-P5-MPL 595 5703 plusmn 1318 2357Lip-DOPE-P5 70 6910 plusmn 1190 5416Lip-DOPE-P5-MPL Indefinable 17545 plusmn 3116 8041

Denotes significant difference from all other formulationsa Median survival timeb Time to reach end pointc Tumor growth delay

58 S Shariat et alCancer Letters 355 (2014) 54ndash60

results have shown the pair of MPL and DOPE used in liposomalformulation had synergic effect on promoting vaccine efficacy whileusing one of them individually in the lipid carrier (Lip-P5-MPL orLip-DOPE-P5 formulations) was impotent to induce immune re-sponse Due to the lipidic structure of MPL and DOPE whichfacilitated incorporation to liposomes and the safe use of anionicliposomes containing MPL in humans [13] Lip-DOPE-P5-MPL for-mulation might be superior to vaccine delivery systems such ascationic liposomes containing CpG ODN or LPDs employed for in-duction of CTL response against HER2-derived peptide antigens[1638]

The success of Lip-DOPE-P5-MPL formulation to elicit a robustCTL response against P5 peptide containing just MHC class I epitopesof HER2neu without the use of T-helper epitope can be accordantwith the claim that MPL has the stimulatory action on APCs [1035]Although formulations containing both CTL and T helper epitopesderived from Her2neu antigen in combination with GM-CSF couldinduce effective CTL responses [3940] and confirmed the claim thatCD4 T cell help was required to activate CTLs [4142] our findingsdemonstrated that this requirement might be dependent on howpeptide vaccines were formulated MPL has been shown to becapable of provoking DCs to produce co-stimulatory molecules andsecrete inflammatory cytokines such as IL-6 IFN-γ and IL-12 whichare essential for activation of T cells [103543ndash45] CTL inductiondue to the stimulatory action of TLR agonists on APCs was previ-ously observed about CpG ODN adjuvant [38]

In conclusion our results show that Lip-DOPE-P5-MPL is an ef-fective formulation for the preparation of a peptide-based HER2neu vaccine Liposomes composed of DMPCDMPGCholDOPEcontaining MPL can deliver peptide antigen to the cytosol and induceCTL response that can be used prophylactically to reduce tumorgrowth The benefits of this formulation (easy manufacturing processand safe use in human) can make it a potential candidate to alter-native ones for developing liposomal vaccines in terms of antitumortherapies in breast cancers in which HER2neu antigen overexpresses

Conflict of interest

The authors declare no conflict of interest

Acknowledgements

We are grateful for the financial support from Shahid BeheshtiUniversity of Medical Sciences Biotechnology Research Center andNanotechnology Research Center Mashhad University of MedicalSciences We would also like to thank Azam Abbasi and Zahra Saberifor their excellent technical assistance This study was a part of SShariatrsquos PhD dissertation

References

[1] J Couzin-Frankel Breakthrough of the year 2013 Cancer immunotherapyScience 342 (2013) 1432ndash1433

[2] HL Hanson DL Donermeyer H Ikeda JM White V Shankaran LJ Old et alEradication of established tumors by CD8+ T cell adoptive immunotherapyImmunity 13 (2000) 265ndash276

[3] K Matsui LA OrsquoMara PM Allen Successful elimination of large establishedtumors and avoidance of antigen-loss variants by aggressive adoptive T cellimmunotherapy Int Immunol 15 (2003) 797ndash805

[4] RM Steinman Decisions about dendritic cells past present and future AnnuRev Immunol 30 (2012) 1ndash22

[5] K Palucka J Banchereau Human dendritic cell subsets in vaccination CurrOpin Immunol 25 (2013) 396ndash402

[6] J Parkin B Cohen An overview of the immune system Lancet 357 (2001)1777ndash1789

[7] DS Watson AN Endsley L Huang Design considerations for liposomalvaccines influence of formulation parameters on antibody and cell-mediatedimmune responses to liposome associated antigens Vaccine 30 (2012) 2256ndash2272

[8] CR Alving Liposomes as carriers of antigens and adjuvants J ImmunolMethods 140 (1991) 1ndash13

[9] P Nordly HB Madsen HM Nielsen C Foged Status and future prospects oflipid-based particulate delivery systems as vaccine adjuvants and theircombination with immunostimulators Expert Opin Drug Deliv 6 (2009)657ndash672

[10] JT Ulrich KR Myers Monophosphoryl lipid A as an adjuvant Past experiencesand new directions Pharm Biotechnol 6 (1995) 495ndash524

[11] CR Alving Lipopolysaccharide lipid A and liposomes containing lipid A asimmunologic adjuvants Immunobiology 187 (1993) 430ndash446

[12] CR Casella TC Mitchell Putting endotoxin to work for us monophosphoryllipid A as a safe and effective vaccine adjuvant Cell Mol Life Sci 65 (2008)3231ndash3240

[13] E Vacchelli L Galluzzi A Eggermont WH Fridman J Galon C Sautes-Fridmanet al Trial watch FDA-approved Toll-like receptor agonists for cancer therapyOncoimmunology 1 (2012) 894ndash907

[14] Y Yarden Biology of HER2 and its importance in breast cancer Oncology 61(Suppl 2) (2001) 1ndash13

[15] CN Baxevanis NN Sotiriadou AD Gritzapis PA Sotiropoulou SA Perez NTCacoullos et al Immunogenic HER-2neu peptides as tumor vaccines CancerImmunol Immunother 55 (2006) 85ndash95

[16] SA Jalali M Sankian J Tavakkol-Afshari MR Jaafari Induction of tumor-specific immunity by multi-epitope rat HER2neu-derived peptides encapsulatedin LPD nanoparticles Nanomedicine 8 (2012) 692ndash701

[17] AA Levin A review of the issues in the pharmacokinetics and toxicology ofphosphorothioate antisense oligonucleotides Biochim Biophys Acta 1489(1999) 69ndash84

[18] S Shariat A Badiee MR Jaafari SA Mortazavi Optimization of a method toprepare liposomes containing HER2neu- derived peptide as a vaccine deliverysystem for breast cancer Iran J Pharm Res 13 (2014) 15ndash25

[19] P Harmon D Cabral-Lilly RA Reed FP Maurio JC Franklin A Janoff Therelease and detection of endotoxin from liposomes Anal Biochem 250 (1997)139ndash146

[20] GR Bartlett Phosphorus assay in column chromatography J Biol Chem 234(1959) 466ndash468

[21] R Lichtenfels WE Biddison H Schulz AB Vogt R Martin CARE-LASS(calcein-release-assay) an improved fluorescence-based test system tomeasure cytotoxic T lymphocyte activity J Immunol Methods 172 (1994)227ndash239

[22] Z Huang MR Jaafari FC Szoka Jr Disterolphospholipids nonexchangeablelipids and their application to liposomal drug delivery Angew Chem Int EdEngl 48 (2009) 4146ndash4149

[23] T Schluep J Hwang J Cheng JD Heidel DW Bartlett B Hollister et alPreclinical efficacy of the camptothecin-polymer conjugate IT-101 in multiplecancer models Clin Cancer Res 12 (2006) 1606ndash1614

[24] MF Bachmann GT Jennings Vaccine delivery a matter of size geometrykinetics and molecular patterns Nat Rev Immunol 10 (2010) 787ndash796

[25] AH Hale H-2 antigens incorporated into phospholipid vesicles elicit specificallogeneic cytotoxic T lymphocytes Cell Immunol 55 (1980) 328ndash341

[26] L Raphael BH Tom Liposome facilitated xenogeneic approach for studyinghuman colon cancer immunity carrier and adjuvant effect of liposomes ClinExp Immunol 55 (1984) 1ndash13

[27] RM Steinman L Bonifaz S Fujii K Liu D Bonnyay S Yamazaki et al Theinnate functions of dendritic cells in peripheral lymphoid tissues Adv Exp MedBiol 560 (2005) 83ndash97

[28] R Reddy F Zhou L Huang F Carbone M Bevan BT Rouse pH sensitiveliposomes provide an efficient means of sensitizing target cells to class Irestricted CTL recognition of a soluble protein J Immunol Methods 141 (1991)157ndash163

[29] S Nair F Zhou R Reddy L Huang BT Rouse Soluble proteins delivered todendritic cells via pH-sensitive liposomes induce primary cytotoxic Tlymphocyte responses in vitro J Exp Med 175 (1992) 609ndash612

[30] JS Chang MJ Choi HS Cheong K Kim Development of Th1-mediated CD8+effector T cells by vaccination with epitope peptides encapsulated in pH-sensitive liposomes Vaccine 19 (2001) 3608ndash3614

[31] L Luo Y Li JS Chang SY Cho TY Kim MJ Choi et al Induction of V3-specificcytotoxic T lymphocyte responses by HIV gag particles carrying multipleimmunodominant V3 epitopes of gp120 Virology 240 (1998) 316ndash325

[32] DP Siegel RM Epand The mechanism of lamellar-to-inverted hexagonal phasetransitions in phosphatidylethanolamine implications for membrane fusionmechanisms Biophys J 73 (1997) 3089ndash3111

[33] J Connor MB Yatvin L Huang pH-sensitive liposomes acid-induced liposomefusion Proc Natl Acad Sci USA 81 (1984) 1715ndash1718

[34] N Duumlzguumlnes RM Straubinger PA Baldwin DS Friend D PapahadjopoulosProton-induced fusion of oleic acid-phosphatidylethanolamine liposomesBiochemistry 24 (1985) 3091ndash3098

[35] DJ Marciani Vaccine adjuvants role and mechanisms of action in vaccineimmunogenicity Drug Discov Today 8 (2003) 934ndash943

[36] B Guy The perfect mix recent progress in adjuvant research Nat RevMicrobiol 5 (2007) 505ndash517

[37] VE Schijns EC Lavelle Trends in vaccine adjuvants Expert Rev Vaccines 10(2011) 539ndash550

[38] WM Li WH Dragowska MB Bally MP Schutze-Redelmeier Effectiveinduction of CD8+ T-cell response using CpG oligodeoxynucleotides andHER-2neu-derived peptide co-encapsulated in liposomes Vaccine 21 (2003)3319ndash3329

59S Shariat et alCancer Letters 355 (2014) 54ndash60

cytotoxicity activity induced by Lip-DOPE-P5-MPL was specificagainst rHER2neu CTL response was not observed against rHER2neu-expressing negative CT26 cells (Fig 4)

Anti tumor effects of Lip-DOPE-P5-MPL vaccination in BALBc

Lip-DOPE-P5-MPL formulation had a superior tumor growth in-hibition in the TUBO tumor mice model (Fig 5A) The survival timewas also significantly prolonged in mice following Lip-DOPE-P5-MPL vaccination compared to the other formulations (Fig 5B) Sincenone of the mice vaccinated with Lip-DOPE-P5-MPL died during theexperiment median survival was indefinable in this group Mediansurvival time (MST) as well as TTE and TGD for each treatmentgroup are summarized in Table 3

Discussion

In the present study we attempted to enhance immunogenic-ity and adjuvanicity of P5 peptide a synthetic peptide containingCTL multi-epitope from rHER2neu protein by conjugating with

different liposomal formulations The results showed P5 peptide con-jugating with liposomes alone (Lip-P5) was incapable of inducingany immune response Although some studies reported liposomesalone could elicit CTL response against encapsulated peptide anti-gens [2526] this formulation could have been used exclusively asa carrier for antigen delivery with no potential to conduct anti-gens into the cytosol Lip-P5 formulation had a particle size of 110ndash150 nm that is needed for efficiently draining to lymph nodes whereCD8+ lymphoid DCs are present [2427]

Inclusion of a pH sensitive lipid like DOPE in the liposome struc-ture has been frequently demonstrated as an efficient strategy tointroduce antigens into MHC class I pathway [28ndash31] After endo-cytosis at the pH of the endosomal compartment the transition ofDOPE from lamellar to hexagonal phase occurs It induces the fusionof liposomes with the endosomal membrane and subsequently li-posomal antigens are released toward the cytosol [32ndash34] Howeveras results have shown inclusion of DOPE alone in liposomal for-mulation (Lip-DOPE-P5) was not sufficient to elicit an effectiveimmune response Lip-DOPE-P5 formulation might have been ableto deliver P5 antigen to MHC class I molecules successfully howeveronce the peptide antigen was presented to CD8+ lymphocytes byAPCs presence of co-stimulatory molecules on the APCs was alsorequired for activation of CD8 cells to produce CTLs [35ndash37] MPLcan induce intracellular signaling pathways leading to productionof these co-stimulatory molecules through TLR4 stimulation [35]Co-formulation of MPL and DOPE in liposomes (Lip-DOPE-P5-MPL) induced an effective cellular immune response characterizedwith the higher IFN-γ producing CD8+ cells and CTL activity As

Fig 4 Antigen-specific CTL response induced by various formulations at two dif-ferent ratios of effector to target cells (ET) was assessed using an in vitro CTL activityassay Splenocytes isolated from mice (four in each group) were incubated with CalceinAM-loaded rHER2neu-expressing TUBO tumor cells and rHER2neu-expressing neg-ative CT26 cells (see method)The data indicate the mean plusmn SEM (n = 4) denotessignificant difference from all other formulations at both various effector-to-targetratios

A B

Fig 5 Protective effects of vaccination with different formulations in BALBc mice against a TUBO tumor model (A) Immunized mice (six in each group) were challenged14 days post last booster with 5 times 105 TUBO cells Tumor size was calculated based on three dimensions The values are means of tumor size plusmn SEM (n = 6) (B) Effects ofimmunization on survival time were monitored for a period of 84 days among BALBc mice (n = 6) denotes significant difference from all other formulations

Table 3Therapeutic efficacy data of different liposomal vaccine formulations in TUBO tumormice model (n = 6)

Formulation MSTa (day) TTEb (day plusmn SD) TGDc

Buffer 525 4797 plusmn 881 ndashP5 56 5567 plusmn 1344 1858Lip-DOPE-MPL 63 5803 plusmn 836 3190Lip-P5 665 6267 plusmn 1254 3531Lip-P5-MPL 595 5703 plusmn 1318 2357Lip-DOPE-P5 70 6910 plusmn 1190 5416Lip-DOPE-P5-MPL Indefinable 17545 plusmn 3116 8041

Denotes significant difference from all other formulationsa Median survival timeb Time to reach end pointc Tumor growth delay

58 S Shariat et alCancer Letters 355 (2014) 54ndash60

results have shown the pair of MPL and DOPE used in liposomalformulation had synergic effect on promoting vaccine efficacy whileusing one of them individually in the lipid carrier (Lip-P5-MPL orLip-DOPE-P5 formulations) was impotent to induce immune re-sponse Due to the lipidic structure of MPL and DOPE whichfacilitated incorporation to liposomes and the safe use of anionicliposomes containing MPL in humans [13] Lip-DOPE-P5-MPL for-mulation might be superior to vaccine delivery systems such ascationic liposomes containing CpG ODN or LPDs employed for in-duction of CTL response against HER2-derived peptide antigens[1638]

The success of Lip-DOPE-P5-MPL formulation to elicit a robustCTL response against P5 peptide containing just MHC class I epitopesof HER2neu without the use of T-helper epitope can be accordantwith the claim that MPL has the stimulatory action on APCs [1035]Although formulations containing both CTL and T helper epitopesderived from Her2neu antigen in combination with GM-CSF couldinduce effective CTL responses [3940] and confirmed the claim thatCD4 T cell help was required to activate CTLs [4142] our findingsdemonstrated that this requirement might be dependent on howpeptide vaccines were formulated MPL has been shown to becapable of provoking DCs to produce co-stimulatory molecules andsecrete inflammatory cytokines such as IL-6 IFN-γ and IL-12 whichare essential for activation of T cells [103543ndash45] CTL inductiondue to the stimulatory action of TLR agonists on APCs was previ-ously observed about CpG ODN adjuvant [38]

In conclusion our results show that Lip-DOPE-P5-MPL is an ef-fective formulation for the preparation of a peptide-based HER2neu vaccine Liposomes composed of DMPCDMPGCholDOPEcontaining MPL can deliver peptide antigen to the cytosol and induceCTL response that can be used prophylactically to reduce tumorgrowth The benefits of this formulation (easy manufacturing processand safe use in human) can make it a potential candidate to alter-native ones for developing liposomal vaccines in terms of antitumortherapies in breast cancers in which HER2neu antigen overexpresses

Conflict of interest

The authors declare no conflict of interest

Acknowledgements

We are grateful for the financial support from Shahid BeheshtiUniversity of Medical Sciences Biotechnology Research Center andNanotechnology Research Center Mashhad University of MedicalSciences We would also like to thank Azam Abbasi and Zahra Saberifor their excellent technical assistance This study was a part of SShariatrsquos PhD dissertation

References

[1] J Couzin-Frankel Breakthrough of the year 2013 Cancer immunotherapyScience 342 (2013) 1432ndash1433

[2] HL Hanson DL Donermeyer H Ikeda JM White V Shankaran LJ Old et alEradication of established tumors by CD8+ T cell adoptive immunotherapyImmunity 13 (2000) 265ndash276

[3] K Matsui LA OrsquoMara PM Allen Successful elimination of large establishedtumors and avoidance of antigen-loss variants by aggressive adoptive T cellimmunotherapy Int Immunol 15 (2003) 797ndash805

[4] RM Steinman Decisions about dendritic cells past present and future AnnuRev Immunol 30 (2012) 1ndash22

[5] K Palucka J Banchereau Human dendritic cell subsets in vaccination CurrOpin Immunol 25 (2013) 396ndash402

[6] J Parkin B Cohen An overview of the immune system Lancet 357 (2001)1777ndash1789

[7] DS Watson AN Endsley L Huang Design considerations for liposomalvaccines influence of formulation parameters on antibody and cell-mediatedimmune responses to liposome associated antigens Vaccine 30 (2012) 2256ndash2272

[8] CR Alving Liposomes as carriers of antigens and adjuvants J ImmunolMethods 140 (1991) 1ndash13

[9] P Nordly HB Madsen HM Nielsen C Foged Status and future prospects oflipid-based particulate delivery systems as vaccine adjuvants and theircombination with immunostimulators Expert Opin Drug Deliv 6 (2009)657ndash672

[10] JT Ulrich KR Myers Monophosphoryl lipid A as an adjuvant Past experiencesand new directions Pharm Biotechnol 6 (1995) 495ndash524

[11] CR Alving Lipopolysaccharide lipid A and liposomes containing lipid A asimmunologic adjuvants Immunobiology 187 (1993) 430ndash446

[12] CR Casella TC Mitchell Putting endotoxin to work for us monophosphoryllipid A as a safe and effective vaccine adjuvant Cell Mol Life Sci 65 (2008)3231ndash3240

[13] E Vacchelli L Galluzzi A Eggermont WH Fridman J Galon C Sautes-Fridmanet al Trial watch FDA-approved Toll-like receptor agonists for cancer therapyOncoimmunology 1 (2012) 894ndash907

[14] Y Yarden Biology of HER2 and its importance in breast cancer Oncology 61(Suppl 2) (2001) 1ndash13

[15] CN Baxevanis NN Sotiriadou AD Gritzapis PA Sotiropoulou SA Perez NTCacoullos et al Immunogenic HER-2neu peptides as tumor vaccines CancerImmunol Immunother 55 (2006) 85ndash95

[16] SA Jalali M Sankian J Tavakkol-Afshari MR Jaafari Induction of tumor-specific immunity by multi-epitope rat HER2neu-derived peptides encapsulatedin LPD nanoparticles Nanomedicine 8 (2012) 692ndash701

[17] AA Levin A review of the issues in the pharmacokinetics and toxicology ofphosphorothioate antisense oligonucleotides Biochim Biophys Acta 1489(1999) 69ndash84

[18] S Shariat A Badiee MR Jaafari SA Mortazavi Optimization of a method toprepare liposomes containing HER2neu- derived peptide as a vaccine deliverysystem for breast cancer Iran J Pharm Res 13 (2014) 15ndash25

[19] P Harmon D Cabral-Lilly RA Reed FP Maurio JC Franklin A Janoff Therelease and detection of endotoxin from liposomes Anal Biochem 250 (1997)139ndash146

[20] GR Bartlett Phosphorus assay in column chromatography J Biol Chem 234(1959) 466ndash468

[21] R Lichtenfels WE Biddison H Schulz AB Vogt R Martin CARE-LASS(calcein-release-assay) an improved fluorescence-based test system tomeasure cytotoxic T lymphocyte activity J Immunol Methods 172 (1994)227ndash239

[22] Z Huang MR Jaafari FC Szoka Jr Disterolphospholipids nonexchangeablelipids and their application to liposomal drug delivery Angew Chem Int EdEngl 48 (2009) 4146ndash4149

[23] T Schluep J Hwang J Cheng JD Heidel DW Bartlett B Hollister et alPreclinical efficacy of the camptothecin-polymer conjugate IT-101 in multiplecancer models Clin Cancer Res 12 (2006) 1606ndash1614

[24] MF Bachmann GT Jennings Vaccine delivery a matter of size geometrykinetics and molecular patterns Nat Rev Immunol 10 (2010) 787ndash796

[25] AH Hale H-2 antigens incorporated into phospholipid vesicles elicit specificallogeneic cytotoxic T lymphocytes Cell Immunol 55 (1980) 328ndash341

[26] L Raphael BH Tom Liposome facilitated xenogeneic approach for studyinghuman colon cancer immunity carrier and adjuvant effect of liposomes ClinExp Immunol 55 (1984) 1ndash13

[27] RM Steinman L Bonifaz S Fujii K Liu D Bonnyay S Yamazaki et al Theinnate functions of dendritic cells in peripheral lymphoid tissues Adv Exp MedBiol 560 (2005) 83ndash97

[28] R Reddy F Zhou L Huang F Carbone M Bevan BT Rouse pH sensitiveliposomes provide an efficient means of sensitizing target cells to class Irestricted CTL recognition of a soluble protein J Immunol Methods 141 (1991)157ndash163

[29] S Nair F Zhou R Reddy L Huang BT Rouse Soluble proteins delivered todendritic cells via pH-sensitive liposomes induce primary cytotoxic Tlymphocyte responses in vitro J Exp Med 175 (1992) 609ndash612

[30] JS Chang MJ Choi HS Cheong K Kim Development of Th1-mediated CD8+effector T cells by vaccination with epitope peptides encapsulated in pH-sensitive liposomes Vaccine 19 (2001) 3608ndash3614

[31] L Luo Y Li JS Chang SY Cho TY Kim MJ Choi et al Induction of V3-specificcytotoxic T lymphocyte responses by HIV gag particles carrying multipleimmunodominant V3 epitopes of gp120 Virology 240 (1998) 316ndash325

[32] DP Siegel RM Epand The mechanism of lamellar-to-inverted hexagonal phasetransitions in phosphatidylethanolamine implications for membrane fusionmechanisms Biophys J 73 (1997) 3089ndash3111

[33] J Connor MB Yatvin L Huang pH-sensitive liposomes acid-induced liposomefusion Proc Natl Acad Sci USA 81 (1984) 1715ndash1718

[34] N Duumlzguumlnes RM Straubinger PA Baldwin DS Friend D PapahadjopoulosProton-induced fusion of oleic acid-phosphatidylethanolamine liposomesBiochemistry 24 (1985) 3091ndash3098

[35] DJ Marciani Vaccine adjuvants role and mechanisms of action in vaccineimmunogenicity Drug Discov Today 8 (2003) 934ndash943

[36] B Guy The perfect mix recent progress in adjuvant research Nat RevMicrobiol 5 (2007) 505ndash517

[37] VE Schijns EC Lavelle Trends in vaccine adjuvants Expert Rev Vaccines 10(2011) 539ndash550

[38] WM Li WH Dragowska MB Bally MP Schutze-Redelmeier Effectiveinduction of CD8+ T-cell response using CpG oligodeoxynucleotides andHER-2neu-derived peptide co-encapsulated in liposomes Vaccine 21 (2003)3319ndash3329

59S Shariat et alCancer Letters 355 (2014) 54ndash60

results have shown the pair of MPL and DOPE used in liposomalformulation had synergic effect on promoting vaccine efficacy whileusing one of them individually in the lipid carrier (Lip-P5-MPL orLip-DOPE-P5 formulations) was impotent to induce immune re-sponse Due to the lipidic structure of MPL and DOPE whichfacilitated incorporation to liposomes and the safe use of anionicliposomes containing MPL in humans [13] Lip-DOPE-P5-MPL for-mulation might be superior to vaccine delivery systems such ascationic liposomes containing CpG ODN or LPDs employed for in-duction of CTL response against HER2-derived peptide antigens[1638]

The success of Lip-DOPE-P5-MPL formulation to elicit a robustCTL response against P5 peptide containing just MHC class I epitopesof HER2neu without the use of T-helper epitope can be accordantwith the claim that MPL has the stimulatory action on APCs [1035]Although formulations containing both CTL and T helper epitopesderived from Her2neu antigen in combination with GM-CSF couldinduce effective CTL responses [3940] and confirmed the claim thatCD4 T cell help was required to activate CTLs [4142] our findingsdemonstrated that this requirement might be dependent on howpeptide vaccines were formulated MPL has been shown to becapable of provoking DCs to produce co-stimulatory molecules andsecrete inflammatory cytokines such as IL-6 IFN-γ and IL-12 whichare essential for activation of T cells [103543ndash45] CTL inductiondue to the stimulatory action of TLR agonists on APCs was previ-ously observed about CpG ODN adjuvant [38]

In conclusion our results show that Lip-DOPE-P5-MPL is an ef-fective formulation for the preparation of a peptide-based HER2neu vaccine Liposomes composed of DMPCDMPGCholDOPEcontaining MPL can deliver peptide antigen to the cytosol and induceCTL response that can be used prophylactically to reduce tumorgrowth The benefits of this formulation (easy manufacturing processand safe use in human) can make it a potential candidate to alter-native ones for developing liposomal vaccines in terms of antitumortherapies in breast cancers in which HER2neu antigen overexpresses

Conflict of interest

The authors declare no conflict of interest

Acknowledgements

We are grateful for the financial support from Shahid BeheshtiUniversity of Medical Sciences Biotechnology Research Center andNanotechnology Research Center Mashhad University of MedicalSciences We would also like to thank Azam Abbasi and Zahra Saberifor their excellent technical assistance This study was a part of SShariatrsquos PhD dissertation

References

[1] J Couzin-Frankel Breakthrough of the year 2013 Cancer immunotherapyScience 342 (2013) 1432ndash1433

[2] HL Hanson DL Donermeyer H Ikeda JM White V Shankaran LJ Old et alEradication of established tumors by CD8+ T cell adoptive immunotherapyImmunity 13 (2000) 265ndash276

[3] K Matsui LA OrsquoMara PM Allen Successful elimination of large establishedtumors and avoidance of antigen-loss variants by aggressive adoptive T cellimmunotherapy Int Immunol 15 (2003) 797ndash805

[4] RM Steinman Decisions about dendritic cells past present and future AnnuRev Immunol 30 (2012) 1ndash22

[5] K Palucka J Banchereau Human dendritic cell subsets in vaccination CurrOpin Immunol 25 (2013) 396ndash402

[6] J Parkin B Cohen An overview of the immune system Lancet 357 (2001)1777ndash1789

[7] DS Watson AN Endsley L Huang Design considerations for liposomalvaccines influence of formulation parameters on antibody and cell-mediatedimmune responses to liposome associated antigens Vaccine 30 (2012) 2256ndash2272

[8] CR Alving Liposomes as carriers of antigens and adjuvants J ImmunolMethods 140 (1991) 1ndash13

[9] P Nordly HB Madsen HM Nielsen C Foged Status and future prospects oflipid-based particulate delivery systems as vaccine adjuvants and theircombination with immunostimulators Expert Opin Drug Deliv 6 (2009)657ndash672

[10] JT Ulrich KR Myers Monophosphoryl lipid A as an adjuvant Past experiencesand new directions Pharm Biotechnol 6 (1995) 495ndash524

[11] CR Alving Lipopolysaccharide lipid A and liposomes containing lipid A asimmunologic adjuvants Immunobiology 187 (1993) 430ndash446

[12] CR Casella TC Mitchell Putting endotoxin to work for us monophosphoryllipid A as a safe and effective vaccine adjuvant Cell Mol Life Sci 65 (2008)3231ndash3240

[13] E Vacchelli L Galluzzi A Eggermont WH Fridman J Galon C Sautes-Fridmanet al Trial watch FDA-approved Toll-like receptor agonists for cancer therapyOncoimmunology 1 (2012) 894ndash907

[14] Y Yarden Biology of HER2 and its importance in breast cancer Oncology 61(Suppl 2) (2001) 1ndash13

[15] CN Baxevanis NN Sotiriadou AD Gritzapis PA Sotiropoulou SA Perez NTCacoullos et al Immunogenic HER-2neu peptides as tumor vaccines CancerImmunol Immunother 55 (2006) 85ndash95

[16] SA Jalali M Sankian J Tavakkol-Afshari MR Jaafari Induction of tumor-specific immunity by multi-epitope rat HER2neu-derived peptides encapsulatedin LPD nanoparticles Nanomedicine 8 (2012) 692ndash701

[17] AA Levin A review of the issues in the pharmacokinetics and toxicology ofphosphorothioate antisense oligonucleotides Biochim Biophys Acta 1489(1999) 69ndash84

[18] S Shariat A Badiee MR Jaafari SA Mortazavi Optimization of a method toprepare liposomes containing HER2neu- derived peptide as a vaccine deliverysystem for breast cancer Iran J Pharm Res 13 (2014) 15ndash25

[19] P Harmon D Cabral-Lilly RA Reed FP Maurio JC Franklin A Janoff Therelease and detection of endotoxin from liposomes Anal Biochem 250 (1997)139ndash146

[20] GR Bartlett Phosphorus assay in column chromatography J Biol Chem 234(1959) 466ndash468

[21] R Lichtenfels WE Biddison H Schulz AB Vogt R Martin CARE-LASS(calcein-release-assay) an improved fluorescence-based test system tomeasure cytotoxic T lymphocyte activity J Immunol Methods 172 (1994)227ndash239

[22] Z Huang MR Jaafari FC Szoka Jr Disterolphospholipids nonexchangeablelipids and their application to liposomal drug delivery Angew Chem Int EdEngl 48 (2009) 4146ndash4149

[23] T Schluep J Hwang J Cheng JD Heidel DW Bartlett B Hollister et alPreclinical efficacy of the camptothecin-polymer conjugate IT-101 in multiplecancer models Clin Cancer Res 12 (2006) 1606ndash1614

[24] MF Bachmann GT Jennings Vaccine delivery a matter of size geometrykinetics and molecular patterns Nat Rev Immunol 10 (2010) 787ndash796

[25] AH Hale H-2 antigens incorporated into phospholipid vesicles elicit specificallogeneic cytotoxic T lymphocytes Cell Immunol 55 (1980) 328ndash341

[26] L Raphael BH Tom Liposome facilitated xenogeneic approach for studyinghuman colon cancer immunity carrier and adjuvant effect of liposomes ClinExp Immunol 55 (1984) 1ndash13

[27] RM Steinman L Bonifaz S Fujii K Liu D Bonnyay S Yamazaki et al Theinnate functions of dendritic cells in peripheral lymphoid tissues Adv Exp MedBiol 560 (2005) 83ndash97

[28] R Reddy F Zhou L Huang F Carbone M Bevan BT Rouse pH sensitiveliposomes provide an efficient means of sensitizing target cells to class Irestricted CTL recognition of a soluble protein J Immunol Methods 141 (1991)157ndash163

[29] S Nair F Zhou R Reddy L Huang BT Rouse Soluble proteins delivered todendritic cells via pH-sensitive liposomes induce primary cytotoxic Tlymphocyte responses in vitro J Exp Med 175 (1992) 609ndash612

[30] JS Chang MJ Choi HS Cheong K Kim Development of Th1-mediated CD8+effector T cells by vaccination with epitope peptides encapsulated in pH-sensitive liposomes Vaccine 19 (2001) 3608ndash3614

[31] L Luo Y Li JS Chang SY Cho TY Kim MJ Choi et al Induction of V3-specificcytotoxic T lymphocyte responses by HIV gag particles carrying multipleimmunodominant V3 epitopes of gp120 Virology 240 (1998) 316ndash325

[32] DP Siegel RM Epand The mechanism of lamellar-to-inverted hexagonal phasetransitions in phosphatidylethanolamine implications for membrane fusionmechanisms Biophys J 73 (1997) 3089ndash3111

[33] J Connor MB Yatvin L Huang pH-sensitive liposomes acid-induced liposomefusion Proc Natl Acad Sci USA 81 (1984) 1715ndash1718

[34] N Duumlzguumlnes RM Straubinger PA Baldwin DS Friend D PapahadjopoulosProton-induced fusion of oleic acid-phosphatidylethanolamine liposomesBiochemistry 24 (1985) 3091ndash3098

[35] DJ Marciani Vaccine adjuvants role and mechanisms of action in vaccineimmunogenicity Drug Discov Today 8 (2003) 934ndash943

[36] B Guy The perfect mix recent progress in adjuvant research Nat RevMicrobiol 5 (2007) 505ndash517

[37] VE Schijns EC Lavelle Trends in vaccine adjuvants Expert Rev Vaccines 10(2011) 539ndash550

[38] WM Li WH Dragowska MB Bally MP Schutze-Redelmeier Effectiveinduction of CD8+ T-cell response using CpG oligodeoxynucleotides andHER-2neu-derived peptide co-encapsulated in liposomes Vaccine 21 (2003)3319ndash3329

59S Shariat et alCancer Letters 355 (2014) 54ndash60

[39] ML Disis KH Grabstein PR Sleath MA Cheever Generation of immunityto the HER-2neu oncogenic protein in patients with breast and ovarian cancerusing a peptide-based vaccine Clin Cancer Res 5 (1999) 1289ndash1297

[40] KL Knutson K Schiffman ML Disis Immunization with a HER-2neu helperpeptide vaccine generates HER-2neu CD8 T-cell immunity in cancer patientsJ Clin Invest 107 (2001) 477ndash484

[41] S Crotty R Ahmed Immunological memory in humans Semin Immunol 16(2004) 197ndash203

[42] FG Gao V Khammanivong WJ Liu GR Leggatt IH Frazer GJ FernandoAntigen-specific CD4+ T-cell help is required to activate a memoryCD8+ T cell to a fully functional tumor killer cell Cancer Res 62 (2002)6438ndash6441

[43] JR Baldridge P McGowan JT Evans C Cluff S Mossman D Johnson et alTaking a Toll on human disease toll-like receptor 4 agonists as vaccine adjuvantsand monotherapeutic agents Expert Opin Biol Ther 4 (2004) 1129ndash1138

[44] G De Becker V Moulin B Pajak C Bruck M Francotte C Thiriart et al Theadjuvant monophosphoryl lipid A increases the function of antigen-presentingcells Int Immunol 12 (2000) 807ndash815

[45] S Mazumder M Maji N Ali Potentiating effects of MPL on DSPC bearingcationic liposomes promote recombinant GP63 vaccine efficacy highimmunogenicity and protection PLoS Negl Trop Dis 5 (2011) e1429

60 S Shariat et alCancer Letters 355 (2014) 54ndash60