utilizing 
 the patient s immune system represents a promising 
 approach for eradication of cancer .
the immune 
 system is well equipped to identify and eliminate malignant cells , 
 but cancer cells can escape from immune surveillance , which can lead 
 to the development of tumors with the potential to metastasize .
cancer vaccines aim to evoke a strong adaptive immune response against 
 tumor antigens , and among many different strategies applied in recent 
 years , synthetic peptide epitopes have been an important approach 
 for t cell based immunotherapy of cancer .
however , unsatisfactory antitumor effects in both animal models 
 and humans have raised the question whether these peptide epitopes , 
 even in combination with strong adjuvants , are sufficiently immunogenic .
insufficient efficiency of minimal peptide - based vaccines may be 
 due to their rapid extracellular degradation , fast diffusion from 
 the site of injection and poor uptake by antigen - presenting cells , 
 including dendritic cells ( dcs ) , as well as lack of generation of 
 costimulatory signals . in some publications
, short peptide 
 vaccines have been reported to induce t - cell tolerance and anergy 
 by direct binding to major histocompatibility complex ( mhc ) molecules 
 on all cells , instead of only antigen presenting cells .
therefore , 
 synthetic long peptides ( slps ) have been proposed as a solution of 
 these drawbacks of the minimal peptide epitope vaccines .
however , slps are still prone to enzymatic degradation 
 by proteases present in blood and also by endopeptidases and exopeptidases 
 present on the surface of dcs .
moreover , 
 due to their relatively small size , these peptide vaccines are rapidly 
 cleared from the injection site , and as a consequence , their uptake 
 by dcs is limited .
furthermore , lack of information 
 on pharmacokinetic and physical properties ( solubility / aggregation ) 
 of peptide vaccines impedes control and prediction of immune responses .
numerous studies have demonstrated that delivery 
 of peptide vaccines through particulate carriers ( e.g. , polymeric 
 or lipidic nanostructures ) can improve the efficacy of peptide epitopes 
 through , for example , protection against degradation and better uptake 
 by dcs .
self - assembling peptides have gained increasing attention 
 as a 
 way to construct biomaterials for biomedical applications such as 
 scaffolds for tissue engineering or nanomaterials for drug and vaccine 
 delivery .
self - assembling peptides can 
 be synthesized by both standardized solid phase peptide synthesis 
 methods and recombinant technology .
furthermore , 
 these peptides are easy to formulate since simple dispersion in aqueous 
 media results in the spontaneous formation of discrete and uniform 
 particles , without the need for laborious processing as often the 
 case for other particulate systems .
furthermore , 
 other functionalities such as adjuvanticity and receptor / cell specificity 
 can be introduced in a single polypeptide . in the present study , we have designed short amphiphilic peptides 
 ( sa2 and sa7 : ac - aavvlllwe27-cooh ) that form nanovesicular 
 structures in aqueous solution .
the self - assembly 
 was due to both hydrophobic clustering and intermolecular hydrogen 
 bond formation as driving forces .
progress 
 in polymer science has resulted in a variety of polymers 
 suitable for drug delivery applications .
particularly , stimuli - responsive polymers that respond to certain 
 stimuli such as ph , temperature , light , magnetism , or ultrasound have 
 received tremendous interest in the last decades . as an example , poly(n - isopropylacrylamide ) 
 ( pnipam ) , is hydrophilic and water - soluble below the so - called lower 
 critical solution temperature ( lcst ) , whereas above this temperature 
 of 33 c it is insoluble in water and precipitates . because 
 of
the aim of this study was 
 to augment the immunogenicity of peptide 
 epitopes by conjugating them to self - assembling moieties to obtain 
 well - defined nanostructures for cancer immunotherapy . in the present 
 study ,
the ova250264 peptide ( sgleqlesiinfekl ( covering 
 a cd8 epitope of ovalbumin : siinfekl ) ) and the hpv16 e74357 of the e7 protein of human papillomavirus type 
 16 ( gqaepdrahynivtf ( covering both a cd8 epitope rahynivtf 
 and a cd4 + epitope pdrahyni ) ) were conjugated to the hydrophobic domain 
 of the sa2 peptide ( ac - aavvlllw - cooh ) or to the thermosensitive polymer 
 ( pnipam ) to trigger peptide self - assembly into discrete nanostructures . 

the peptide vaccines were biophysically characterized , and their capacity 
 to induce or expand antigen - specific cd8 t cell were tested 
 in mice .
finally , in vivo studies in mice were conducted 
 to evaluate the efficacy of sapes that contain the hpv peptide epitope 
 ( hpv sapes ) as a therapeutic vaccine .
acetonitrile ( acn ) , dichloromethane 
 ( dcm ) , dimethylformamide ( dmf ) , methyl - tert - butyl 
 ether ( mtbe ) , n - methyl-2-pyrrolidine ( nmp ) , n - hexane , and diethyl ether were purchased from biosolve 
 bv ( valkenswaard , the netherlands ) .
n - isopropylacrylamide 
 ( nipam ; > 99% ) , n , n - dimethyl acrylamide 
 ( dmam ; > 99% ) , deuterium oxide ( d2o ) , deuterated dimethyl 
 sulfoxide , ( 99.9% , dmso - d6 ) , -bromoisobutyryl 
 bromide , triisopropylsilan , nile red , poly - l - lysine , hexafluoroisopropanol 
 ( hfip ) , and 4-(2-hydroxyethyl ) piperazine-1-ethanesulfonic acid ( hepes ) 
 were purchased from sigma - aldrich ( zwijndrecht , the netherlands ) . 

n , n - diisopropyl - ethylamine ( dipea ) was 
 purchased from biosolve ( valkenswaard , the netherlands ) .
cucl and 
 cucl2 were obtained from sigma - aldrich ( zwijndrecht , the 
 netherlands ) .
g / l ) ( pbs ) was purchased from b. 
 braun ( melsungen , germany ) .
iscove s modified dulbecco s 
 medium ( imdm ) was purchased from lonza ( walkersville , usa ) .
cpg odn 
 1826 ( cpg ) was purchased from invivogen ( cayla , france ) .
pe - labeled h-2d epitope e74957 ( rahynivtf)- and pe - labeled h-2d epitope ova257264 ( siinfekl)-containing tetramers were constructed and used for the 
 analysis of peptide - specific ctl immunity as described earlier .
all other chemicals were of analytical grade , 
 and all aqueous solutions were prepared with millipore milli - q deionized 
 water ( 18.2 m ; millipore milli - q system , billerica , ma ) .
resin - protected 
 peptide epitopes of ovalbumin ( ova250264 : devsgleqlesiinfekl ) 
 and human papilloma virus type 16 ( hpv 16 ) oncoprotein e7 ( hpv16-e74357 : gqaepdrahynivtf ) were ordered from ansynth service 
 bv ( roosendaal , netherlands ) .
the purity of the mentioned peptides 
 as determined by reverse - phase hplc and mass spectrometry was > 90% . 

the characteristics of the other peptides that were used in this study 
 are listed in table 1 .
the peptides were synthesized by standard solid - phase peptide synthesis 
 and delivered with a purity > 95% .
the synthesis of the ova and hpv peptide - atrp macroinitiator is 
 shown in scheme 1 . 

resin - protected peptides ( ova250264 or hpv16 e74357 ) with deprotected n - terminus ( equal to 0.1 mmol 
 of the peptide ) was dispersed in and washed three times with 5 ml 
 of dried dichloromethane .
next , the resin was washed three times with 
 5 ml of nmp . subsequently , 3 equiv of dipea and 2 equiv of 2-bromo - isobutyryl 
 bromide ( relative to nh2 groups ) in nmp were added . 

the 
 kaiser test was done to determine nonreacted 
 free amines , and when this test was negative , the reaction was considered 
 completed .
first , the resin washed three times with nmp 
 and subsequently a solution of tfa / water / triisopropylsilan ( 95/2.5/2.5 
 v / v / v % ) was added and incubated for 4 h at room temperature under 
 vigorous shaking to fully deprotect the peptide .
next , the peptide - initiator 
 product was precipitated in a cold mtbe / n - hexane 
 ( 1/1 ; v / v % ) , separated , and washed two times with mtbe / n - hexane using centrifugation ( 3000  g , 5 min , 
 4 c ) .
the obtained product was dispersed in water and freeze - dried 
 using a christ 
the molecular masses of the synthesized peptide - initiator 
 was determined by electrospray mass spectrometry .
the ova250264- and the hpv16 e74357-poly(nipam ) peptide  polymer 
 conjugates , referred to as ova - pnipam and hpv - pnipam , and hpv16 e74357-poly(dmam ) , referred as to hpv - pdmam , were synthesized 
 using atom transfer radical polymerization ( atrp ) .
the peptide macroinitiator 
 ( 20 mol ) was dissolved in 2 ml 
 of water / acn ( 80:20 v / v % ) in a screw - capped septum vial . in case 
 of ova250264 ,
the peptide was first dissolved in 
 an alkaline solution ( ph 12 ) , and subsequently , the ph was adjusted 
 to ph 8.5 with hcl ( 0.5 m ) . in another screw - capped septum vial ,
15 
 mol of cubr , 0.05 mol of cubr2 , and 5.3 mmol 
 ( 600 mg ) of nipam or 525 mg of dmam were dissolved in 3 ml of water / acn 
 ( 80:20 v / v % ) .
the vials were capped with a septum and the solutions 
 were flushed with n2 .
next , the peptide solution was added 
 to the nipam or dmam solution , flushed with n2 , and placed 
 in an ice bath , under rapid stirring .
the polymerization was initiated 
 by adding 0.1 ml of 0.4 m me6tren solution in water ( degassed 
 by flushing with n2 prior to addition ) , which immediately 
 turned the reaction mixture blue .
periodically , 50 l samples 
 were taken , diluted in air - saturated d2o , and analyzed 
 by h nmr to determine the nipam or dmam conversion .
when the conversion was 70% , the reaction 
 was stopped by flushing air into the reaction mixture .
the crude product 
 was dialyzed ( with dialysis cassettes of 3000 kda mwco ) against water 
 at 4 c for 2 days and subsequently lyophilized . the peptide 
polymer 
 conjugates were characterized by gel permeation chromatography ( gpc ) 
 using mixed - d columns ( polymer laboratories ) , a solution of 10 mm 
 licl in dmf as eluent , a flow of 0.7 ml / min , and a temperature of 
 40 c .
a refractive index detector was used , and peg standards 
 were used for calibration .
all peptide formulations indicated in table 1 ( except ova - sa peptide ) were 
 prepared as 
 follows .
one milligram of the peptide was hydrated with 40 l 
 of 0.2 m naoh solution , and subsequently , phosphate buffered saline 
 ( pbs , 140 mm nacl , 13 mm na2hpo4 , and 2.5 mm 
 nah2po4 , ph 7.4 ) was added in 200 l portions 
 up to 1 ml .
after each step , the peptide dispersion was sonicated 
 in a water bath sonicator ( fisher scientific , loughborough , uk ) for 
 3 min at 30 c . before reaching to final volume , the ph was adjusted 
 to ph 7.4 by dropwise adding 0.2 m hcl . to prepare the ova - sa peptide 
 formulation ,
after solvent evaporation , the obtained peptide film was 
 hydrated in 40 l of 0.2 m naoh , and then similar to other peptide 
 preparations , 1 ml of pbs ( 140 mm nacl , 13 mm na2hpo4 , and 2.5 mm nah2po4 , ph 7.4 ) was added 
 in 200 l portions .
after ph adjustment , some of the peptide 
 formulations were supplemented with cpg ( 20 g per injection ; 
 10 l of a 2 mg / ml of a stock solution of cpg in pbs ) as an 
 adjuvant ( table 2 ) .
the dose of antigen 
 in ova - sa , ova - pnipam , 
 and ova peptide was 40 nmol / injection ( concentration : 200 m ) . the dose of antigen in hpv - sa , 
 hpv - pnipam , 
 hpv - pdmam , and hpv peptide was 60 nmol / injection ( concentration : 300 
 m ) .
peptide - pnipam formulations 
 were prepared by either a rapid heating or a heat shock procedure .
 pnipam conjugates 
 and nonthermosensitive peptide - pdmam , as a nonself - assembling peptide 
polymer 
 conjugate , were dissolved in 10 mm pbs or 20 mm hepes at room temperature . 

subsequently , some of the formulations were supplemented with cpg 
 ( 20 g per injection ; 10 l of 2 mg / ml stock solution 
 of cpg in pbs ) , as an adjuvant ( table 2 ) .
for the heat shock procedure , 100 l of polymer 
 solution ( 1 mg / ml ) with or without cpg was added to 900 l of 
 pbs or hepes heated at 50 c .
next , the self - assembled nanostructures 
 were equilibrated at 37 c for at least 5 min before being analyzed 
 for their physicochemical properties . for the rapid heating procedure , 

an aqueous polymer solution ( 110 mg / ml in pbs or hepes ) was 
 rapidly heated at 50 c and kept at this temperature for 5 min . 

subsequently , the formed nanoassemblies were cooled down to 37 c 
 until further analysis .
the critical aggregation concentration ( cac ) of the ova - sa and 
 hpv - sa vaccines was determined using the nile red assay as described 
 by zhang et al . in short ,
peptide - based 
 sapes were dissolved in pbs ( ph 7.4 ) in concentrations ranging from 
 0.1 to 500 g / ml .
next , 200 l of each dilution was spiked 
 with 0.2 l of nile red solution ( 1.25 mm in ethanol ) .
after 
 overnight incubation at room temperature , fluorescence emission ( ex550/em635 ) 
 was measured by a horiba fluorolog fluorometer ( horiba jobin yvon , 
 longjumeau cedex , france ) .
the fluorescence intensity at 635 nm was 
 plotted against the logarithm of peptide concentrations to determine 
 the cac .
the point of intersection of two linear fits of the plot 
 was considered as the cac value .
the cac of ova - pnipam and hpv - pnipam 
 vaccines was determined by dynamic light scattering ( dls , malvern 
 instruments , malvern , u.k . ) . in detail ,
peptide - pnipam conjugates 
 were dissolved in pbs ( ph 7.4 ) in concentrations ranging from 1 to 
 1000 g / ml . the thermosensitive spaes ( peptide - pnipam ) were 
 formed by the rapid heating method , as described above .
the z - average diameter ( zave , nm ) 
 and light scattering intensity ( lsi ) of the sape dispersion were monitored 
 by dls at 37 c .
the lsi was plotted against the logarithm of 
 the conjugate concentrations , and the point of intersection of two 
 linear fits of the plot was considered as the cac of the peptide - pnipam 
 sapes .
the prepared sape particles in pbs 
 ( ph 7.4 ) were sized using an 
 alv cgs-3 goniometer system ( malvern instruments , malvern , u.k . ) equipped 
 with a jds uniphase 22 mw he  ne laser operating at 632.8 nm , 
 an optical fiber - based detector , a digital lv / lse-5003 correlator . 
 and a temperature controller ( julabo water bath ) .
the dls time correlation 
 was analyzed by alv correlator 3.0 software ( alv , langen , germany ) . 

zeta potential measurement of the peptide particles was performed 
 in 10 mm hepes , ph 7.0 , at 25 c for the peptide  peptide 
 and 37 c for the peptide - pnipam assemblies using a malvern zetasizer 
 nano - z ( malvern instruments , malvern , u.k . ) with universal zen 1002 
  dip  cells and dts ( nano ) software ( version 4.20 ) . 

dts 1235 latex beads ( zeta potential transfer standard , malvern instruments , 
 malvern , u.k . ) were used for calibration .
the morphology 
 and size of the peptide - pnipam sapes were visualized by transmission 
 electron microscopy ( tem ) .
briefly , samples ( 0.1 mg / ml in hepes 10 
 mm ) were soaked onto prewarmed carbon - coated copper grids for 2 min , 
 and excess liquid was removed by a filter paper .
the grids were subjected 
 to negative staining with 2% uranyl acetate ( merck , germany ) for 45 
 s and dried for 10 min at room temperature before acquisition of tem 
 images ( tecnai 10 , philips , the netherlands ) .
atomic force microscopy 
 ( afm ) was applied to visualize the morphology of the peptide - based 
 vaccines .
briefly , 40 l of a 0.02% ( w / v ) polylysine solution 
 in water was placed on a freshly cleaved mica substrate and , after 
 5 min incubation , washed with filtered milli - q water followed by drying 
 under n2 flow .
next , 30 l of the sample was loaded 
 on the treated mica and incubated for 1 min , followed by washing three 
 times with 20 l of filtrated milli - q water to discharge nonattached 
 particles and salts .
scanasyst image mode was used at room temperature 
 with a digital instruments nanoscope v , equipped with silicon nitride 
 cantilevers ( veeco , ny , usa ) .
circular dichroism 
 spectra were obtained by a double beam dsm 1000 cd spectrometer ( online 
 instrument systems , bogart , ga , usa ) using quartz cuvettes with different 
 path length appropriate for the different concentrations .
samples 
 of peptide - based sapes ( at concentration of 200 mm ova - sa and 300 
 mm hpv - sa ) were prepared in pbs ( ph 7.4 , 140 mm nacl ) .
five measurements 
 at 25 c using 1 nm intervals and 1 nm bandwidth at a wavelength 
 range from 185250 nm were performed .
next , the spectrum of 
 the buffer was subtracted from the averaged spectrum of the samples . 
 to normalize for concentration ,
tc-1 
 cells were derived 
 from primary lung epithelial cells of c57bl/6 mice cotransformed with 
 hpv-16 e6 and e7 and c - ha - ras oncogenes .
these cells were cultured at 37 c with 5% co2 in 
 imdm , supplemented with 8% fetal calf serum ( fcs ) ( greiner ) , 400 g / ml 
 of geneticin ( g418 ) ( life technology ) , 2 mm l - glutamine , 
 1 mm sodium pyruvate , 2 mm nonessential amino acid , and 100 iu / ml 
 penicillin . to establish antigen 
 presentation in vivo ,
cd8 t cells were 
 isolated from spleen and lymph nodes of cd90.1 ot - i mice .
t cells 
 were enriched using the cd8 + enrichment kit according to manufacturer s 
 protocol ( bd bioscience ) , and 500,000 cells were injected intravenously 
 into the tail vein of cd90.2 mice ( n = 5/group ) . 

injection 
 with self - assembled or soluble peptide constructs containing ova ctl 
 epitopes ( 40 nmol / injection ) ( table 2 ) .
peripheral blood mononuclear cells ( pbmcs ) were 
 collected at days 3 , 5 , 7 , and 10 after immunization to analyze the 
 expansion of the transferred ova - specific t cells caused by cross 
 presentation of the ova antigen .
after lysis of red blood cells , t 
 cells were stained with fluorescently labeled antibodies for cd8 
 and the ot - i congenic marker cd90.1 ( thy1.1 ) and analyzed by flow 
 cytometry .
t cell expansion is expressed as the number of transferred 
 ( ot - i ) t cells as a percentage of total cd8 t cells . to evaluate the potency of the sape vaccines to induce endogenous 
 specific cd8 t cells , nave c57bl/6 mice ( n = 5 ) were s.c .
pbmcs were collected 10 days after prime vaccination and 5 and 7 days 
 after the boost vaccination . to quantify antigen - specific cd8 t cells by flow cytometry , after lysis of red blood cells , 

groups of mice ( n = 12 ) were challenged 
 in the right flank with 100,000 tc-1 tumor cells . at day 7 , when the 
 tumors were palpable ( tumor size between 0.5 and 10 mm ) , mice were s.c . vaccinated with the vaccine formulations ( table 2 ) .
tumor size was 
 measured two - dimensionally with calipers 23 times per week 
 and followed for approximately 35 days .
mice were sacrificed for ethical reasons when 
 the actual tumor volume reached about 2000 mm or when 
 the tumor became ulcerated . before and after booster vaccination , 
 blood samples were collected via tail vein in heparinized tubes for 
 detecting hpv - specific cd8 t by flow cytometry .
kaplan  meier survival curves were applied , and the differences 
 between survival curves were analyzed by log - rank test ( p < 0.05 was considered statistically significant ) .
expansion of 
 antigen - specific cd8 t cells in the blood of immunized 
 mice after booster vaccination with the sape vaccines was compared 
 to control mice by student t test .
the percentage 
 of ot - i cells on day 5 postimmunization in mice treated with sape 
 vaccines was compared to control group by student t test .
the 
 sa2 peptide was previously designed and studied in depth as a self - assembling 
 peptide forming nanovesicles in aqueous solutions . in the present study , we have exploited the capacity of 
 sa2 hydrophobic domain ( sa : ac - aavvlllw - cooh ) to form nanostructures 
 once conjugated to antigen epitopes . to this end ,
peptides ( ova250264 ( sgleqlesiinfekl ) derived 
 from chicken ovalbumin and containing the h2b restricted mhc class 
 i peptide epitope siinfekl , and hpv74357 ( gqaepdrahynivtf ) derived from human papillomavirus e7 protein 
 and containing the h2b restricted mhc class i peptide epitope rahynivtf ) 
 were fused to the c - terminus of the sa domain ( ova - sa and hpv - sa , table 1 ) . in the selected 
 hpv74357 and ova250264 peptides , 

the presence of the natural flanking amino acids , gqaepd at the n - terminus 
 of the hpv and sgleqle at the n - terminus of ova ctl epitopes , allows 
 proper cleavage and processing of the peptides in dcs .
the ova and hpv peptide macroinitiators 
 were synthesized by coupling an atrp initiator to the n - terminus of 
 the peptides ( scheme 1 ) .
the molecular masses of the peptide - initiators were determined 
 using an electron spray mass spectrometer , and this analysis showed 
 that the atrp initiator was coupled to the n - terminus of the peptides 
 ( data not shown ) .
polymer 
 conjugates were synthesized by atrp using nipam and dmam , as the monomer 
 and the peptide macroinitiators ( scheme 2 ) .
polymer conjugates have pnipam block length of mn  30,000 and 40  000 g / mol and 
 a fixed peptide molecular weight of 1719 g / mol ( ova ) and 1717 g / mol 
 ( hpv ) , respectively .
the hpv - pdmam peptide polymer conjugate had also mn  30  000 g / mol , similar to the 
 peptide - pnipam conjugates .
atrp was used for the synthesis of the 
 peptide - pdmam and peptide - pnipam conjugates since this is an efficient 
 polymerization technique , especially for acrylamides , and results 
 in polymers with a very low polydispersity .
the synthesized peptide - pnipam and peptide - pdmam conjugates had 
 an average molecular weight close to the target value and a very low 
 polydispersity .
it has been shown that pnipam is a thermosensitive 
 polymer with an lcst around 33 c in aqueous solution .
consequently , it is expected that peptide - pnipam 
 conjugate can form self - assemblies with a hydrophilic peptide shell 
 and a hydrophobic pnipam core above the lcst of the pnipam blocks 
 as first described for peg - pnipam .
however , 
 pdmam is a nonthermosensitive / fully water - soluble polymer , and the 
 resulting peptide - pdmam conjugate does , therefore , not self - assemble 
 into nanoparticles and was used as a control formulation in the in vivo tumor experiments .
mn = 
 number - average molar weight ( g / mol ) and mw = weight - average molar weight ( g / mol ) as determined by gpc . to characterize the peptide nanostructures , 

several complementary methods were applied ( table 4 ) . in peptide based systems , similar to the 
 previously reported sa2 peptide , amphiphilic peptide epitopes self - assembled 
 into discrete nanostructures above a threshold concentration , the 
 critical aggregation concentration ( cac ) .
the cac values of the ova - sa 
 and hpv - sa vaccines were both ca .
15 m , which is in the same 
 range as that of sa2 , and indicate that 
 the peptide epitope sequences appended to the c - terminus of the self - assembling 
 sa domain had marginal influence on the cac .
afm revealed that 
 ova - sa particles had a spherical morphology with 
 similar particle size as measured by dls ( figure 1 ) .
the obtained hpv - sa nanoparticles were 
 also analyzed at concentrations used for the in vivo experiments ( 300 m ) , which showed consistent characteristics 
 regarding size and morphology .
dls for the hpv - sa peptide revealed 
 that self - assembling peptide formed nanostructures with a mean hydrodynamic 
 diameter of 85 nm and a pdi of 0.4 ( table 4 ) .
afm analysis of the immobilized nanostructures 
 on mica revealed toroidal structures with a diameter of 100 
 nm in accordance with the size obtained by dls ( figure 1 ) .
the observed concave structures may suggest 
 a hollow , vesicular nature of these nanostructures , similar to the 
 vesicular structures formed by the previously reported sa2 peptide .
although the cac for the different sa based 
 peptides were dictated by the sa tag , the morphology of the nanoparticles , 
 however , was affected by the appended peptide epitope sequences .
negative 
 zeta potential was seen for both sapes at ph 7.4 . the isoelectric 
 point ( pi ) of the ova and hpv peptides are 4.1 and 5.7 , respectively , and therefore , they bear negative charge at 
 ph 7.4 .
afm images of ( a ) ova - sa and ( b ) hpv - sa peptides self - assemblies 
 immobilized on poly - l - lysine coated mica .
the previous study on sa2 peptide demonstrated 
 the ability of this 
 peptide to form a stable beta - sheet structure once integrated into 
 self - assemblies . however , comparison 
 between the secondary structures of hpv - sa and ova - sa with sa2 above 
 their cacs can be indicative in similarity or dissimilarity of self - assembling 
 pathways in these series of peptides .
the cd spectrum of the hpv - sa 
 and ova - sa nanostructures showed a negative peak between 210 and 220 
 nm ( figure s1 ) that is typical for peptides 
 forming -sheets .
this result is 
 in accordance with the obtained secondary structure of previously 
 studied sa2 peptide .
peptide - pnipam 
 ( ova and hpv ) sapes prepared by rapid heating in 
 pbs were characterized for their morphology , size , polydispersity , 
 and zeta potential ( table 4 ) .
ova - pnipam and hpv - pnipam sapes had a mean hydrodynamic 
 diameter of 115 and 196 nm , respectively , with a narrow size distribution 
 ( table 4 ) .
tem analysis 
 showed spherical particles of 2080 nm for both ova - pnipam 
 and hpv - pnipam sapes ( figure 2 ) but no particles for hpv - pdmam conjugate .
the mean hydrodynamic 
 diameters of all nanoparticle formulations , determined by tem , were 
 smaller than those determined by dls . in a nonmonodisperse particle 
 suspension
, the mean hydrodynamic diameter of the particles might 
 be overestimated by dls measurement because of the presence of large 
 particles or small aggregates .
tem analysis of sape 
 nanostructures demonstrates that the majority of the particles are 
 in a range of 2050 nm . however , there are a few larger particles 
 of 200500 nm , which affect the actual average particle size 
 of sape nanostructures as measured by dls .
zeta potential analysis 
 of the peptide - pnipam nanoparticles showed a negative zeta potential 
 for both nanoparticles at ph 7.4 ( table 4 ) , which indicates the presence of negatively 
 charged ova or hpv peptide at the surface of the nanoparticles at 
 ph 7.4 .
from all these results , it can be concluded that these amphiphilic 
 peptide polymer conjugates mainly form micelles .
the cac of ova - pnipam 
 and hpv - pnipam sapes was 0.02 mg / ml , comparable to those of 
 other amphiphilic pnipam block copolymers .
preparation of the peptide - pnipam nanoparticles was relatively easy 
 because of the hydrophilicity of pnipam at ambient temperature ( below 
 the lcst of pnipam , 33 c ) , which allows dissolution of the peptide - pnipam 
 conjugates in an aqueous solution and at room temperature .
next , rapid 
 heating of this solution resulted in small and relatively uniform 
 nanoparticles with a narrow size distribution due to dehydration and 
 self - assembling of the pnipam part of the conjugate .
tem pictures of nanoparticles 
 composed of ( a ) hpv - pnipam and ( b ) 
 ova - pnipam sapes . to assess the 
 t cell activation capacity of the sapes in 
 vivo , ova - specific cd8 t cells ( ot - i )
were injected 
 into c57bl/6 mice 1 day before administration of ova containing sapes . 
 in this way ,
the precursor frequency of ova - specific t cells was artificially 
 elevated to high levels , thereby facilitating the read - out of in vivo antigen processing and presentation and enabling 
 the detection of subtle differences between vaccine formulations in 
 terms of ( cross-)presentation of the ova antigens .
the degree of ot - i 
 t cells expansion as a consequence of in vivo ( cross-)presentation 
 of the ova antigens at various time points after vaccination is shown 
 in figure 3 .
high numbers 
 of ot - i cells were observed in the blood samples peaking at day 5 
 after vaccination with both sape vaccines ( ova - sa and pnipam - ova ) 
 adjuvanted with cpg , which proved stronger to the administration of 
 the ova250264 peptide with cpg ( figure 3 ) ( p < 
 0.05 for pnipam - ova ) . the strong expansion of ot - i cells indicated 
 the capacity of these designed vaccines for cross - presentation , which 
 is necessary for effective induction of cellular immunity .
these results clearly show that sape formulations 
 adjuvanted with cpg are efficiently processed in vivo leading to t cell activation . in vivo ( cross)-presentation 
 of ova antigen to 
 ova - specific cd8 ( ot - i ) t cells .
the percentage of ot - i 
 cells relative to the total number of cd8 t cells in pmbcs 
 of the mice vaccinated with 200 l of indicated peptide formulations 
 ( equal to 40 nmol of ova peptide / injection ) on days 2 , 5 , 7 , and 9 
 postimmunization .
the percentage of ot - i cells on day 5 postimmunization 
 in mice treated with ova - sa + cpg and ova - pnipam + cpg vaccines is 
 compared to that of soluble ova peptide + cpg .
n = 5 mice per group , and data are presented 
 as mean  se .
the development of strong functional cd8 t cells is a necessity for robust antitumor immunity .
as the hpv 
 sapes were further evaluated in vivo for antitumor 
 efficacy , the immunostimulatory potential of the hpv vaccines were 
 investigated for induction of an endogenous t cell response by s.c . 

injection of formulated sapes in a prime - boost regimen into nave 
 c57bl/6 mice ( table 2 ) .
ten days after prime injection and 5 and 7 days after the booster , 
 the induction of specific hpv cd8 t cells in the collected 
 blood samples was analyzed using tetramer staining . for both 
 adjuvanted sapes ( hpv - sa and hpv - pnipam ) , as shown in figure 4 ,
the nanoassemblies were able 
 to induce significantly higher hpv - specific cd8 t cell 
 response in mice after booster vaccination , as compared to the control 
 groups ( p < 0.001 ) .
the response increased from 
 day 5 to 7 after booster immunization ( p < 0.001 ) . 

however , after prime immunization , the mice showed weak immune responses . 
 thus , for such peptide vaccines
, not only an adjuvant is necessary 
 to induce a strong immune response but also a booster vaccination 
 is important to induce strong immunity and antigen - specific memory 
 t cells .
percentage of endogenous hpv 16 e74957-specific 
 cd8 t cells in peripheral blood of mice vaccinated with 
 indicated formulations ( equal to 60 nmol of hpv peptide per injection ) 
 10 days after prime vaccination and 6 or 7 days after booster vaccination . 

hpv - sa + cpg was tested in a different experiment thus presented separately 
 because of different background ( untreated mice ) in facs analysis . 

hpv - specific cd8+t cell expansion after booster vaccination in mice 
 treated with hpv - sa + cpg and hpv - pnipam + cpg vaccines is compared 
 to untreated mice and/or soluble hpv peptide + cpg .
n = 5 mice per group , and data are presented 
 as mean  se . in general , therapeutic 
 cancer vaccines
aim to raise the number 
 of antigen - specific cd8 cytotoxic t lymphocyte cells .
our experiments show efficient endogenous cd8 t cell 
 activation ( hpv vaccines ) indicating that these designed vaccines 
 enable effective cross - presentation , which is obligatory for induction 
 of cellular immunity .
our results also 
 point out that the adjuvant and particulate system have a complementary 
 effect to induce efficient immune responses , in agreement with other 
 studies .
it has been reported that cpg 
 enhanced antigen cross - presentation and improved cd8 t 
 cell responses .
the effect of odns containing 
 unmethylated cytosine - guanine ( cpg ) motifs as toll - like receptor 9 
 ( tlr-9 ) agonist on cellular immunity has been associated with its 
 ability to induce and expand the number of activated dcs .
in addition , 
 it can elevate the expansion of antigen - stimulated t cells by inhibition 
 of activation - induced cell death .
therefore , 
 codelivery of efficient adjuvants such as toll - like receptor ligands 
 with the sapes can improve the efficiency of such formulations .
interestingly , several peptide - based toll - like receptor ligands 
 exist ( such as rs01 , gln - glu - ile - asn - ser - ser - tyr ; rs09 , ala - pro - pro - his - ala - leu - ser ) 
 that can be exploited for incorporation into sapes to improve ctl 
 responses .
codelivery of antigen and 
 adjuvant does not only result in proper dc activation and antigen 
 presentation , but it also significantly lowers systemic toxicity due 
 to reduced systemic exposure . to investigate 
 the potential antitumor efficacy of hpv sape vaccines 
 in a mouse model of hpv - related cancer ,
seven days after tumor inoculation ( when the tumors were palpable ) , 
 the treatment group received hpv sape vaccines adjuvanted with cpg 
 in a prime - boost regimen .
in addition , one group of mice was vaccinated 
 with soluble hpv16 e74357 peptide , without a self - assembling 
 domain , + cpg . as negative controls ,
one group did not receive any 
 formulation , and one group was treated with hpv - pdmam + cpg , a similar 
 structure as hpv - pnipam , without sape forming capacities .
tumor size 
 was monitored over time , and the proportions of mice with a tumor 
 size not exceeding the humane end point ( tumor size of 2000 mm ) are presented in a kaplan  meier plot ( figure 5 ) . both hpv sape vaccines
adjuvanted 
 with cpg strongly delayed the tumor growth and significantly prolonged 
 the overall survival of mice compared with untreated mice ( p < 0.0005 for hpv - pnipam + cpg and p < 0.05 for hpv - sa + cpg ) .
importantly , the hpv - pdmam vaccine 
 did not delay tumor outgrowth , indicating that self - assembly into 
 nanostructures is crucial for efficient vaccine function , and merely 
 elongating the peptide by attaching a long polymer chain did not enhance 
 t cell activating properties .
moreover , 
 development of hpv - associated tumors was not delayed by injection 
 of only hpv16 e74357 peptide + cpg , emphasizing 
 the need for nanoparticle formation of the peptide vaccine for efficient 
 t cell priming , delaying tumor growth .
is 
 associated with the particulate form of peptide vaccines , while the 
 soluble form of peptide vaccine is not effective at all .
seven days after tc-1 tumor inoculation in wild - type c57bl/6 mice , 
 and when tumors were palpable , mice were either left untreated or 
 were s.c . vaccinated in the opposite flank with the indicated formulations 
 ( equal to 60 nmol of hpv peptide per injection ) followed by a boost 
 injection 14 days after the prime dose .
statistical differences between 
 the groups were calculated using a log - rank ( mantel  cox ) test . 

the survival of mice vaccinated with sape vaccines ( hpv - sa and hpv - pnipam ) 
 with cpg was significantly longer compared to the no treatment group 
 ( p < 0.0005 for hpv - pnipam + cpg and p < 0.05 for hpv - sa + cpg ) .
however , there was no significant difference 
 in survival between mice vaccinated with either hpv peptide + cpg 
 or hpv - pdmam + cpg and untreated mice .
numbers in the legends represent 
 median survival in days . * p < 0.05 , * * * p < 0.001 .
n = 12 mice per group , pooled 
 results from two independent experiments . based on these results
, it can be concluded that the soluble 
 form 
 of the peptide vaccine even with a strong adjuvant is not effective . 

however , in the form of a particular structure , it is efficiently 
 processed and presented by dcs to activate effector t cells that delay 
 the tumor growth .
it has been shown that peptide vaccines offer 
 a promising approach 
 to raising specific humoral or cellular immune responses against cancer 
 cells .
various studies proposed to deliver 
 peptide vaccines in a particulate form , which favors innate immune 
 recognition and activation . only a few studies 
 explored peptide self - assembly as a means to present peptide epitopes 
 in a particulate form to the immune system .
for example , collier and 
 co - workers produced a nanofiber vaccine by incorporating a fiberizing 
 self - assembling peptide into the structure of a peptide antigen .
they showed that the self - assembling peptide 
 epitope triggered the formation of high - levels of igg1 , igg2a , and 
 igg3 antibodies against the attached antigen epitope .
most other studies used long stretches of amino acids to 
 drive peptide self - assembly , which impedes solid phase peptide synthesis . in addition , it also increases the risk of immunogenicity against 
 the carrier part . in this study
, we demonstrated 
 that by attaching an eight amino acid peptide sequence or a thermosensitive 
 polymer tail to antigenic peptide epitopes with different length and 
 composition , discrete nanostructures could be generated in a reproducible 
 fashion .
this strategy can overcome some shortages of other particulate 
 systems . besides the ease of production and well - defined formulation , 
 several studies indicated a correlation between epitope density and 
 immune responses .
in the designed sapes , the generated particles comprise 
 a high density of antigenic epitopes displayed on the surface of nanoparticles , 
 while in several studied polymeric nanoparticles the reported peptide 
 loading was on the average < 5% .
our in vivo results have shown that relatively 
 short hpv peptides in particulate forms are able to significantly 
 delay the tumor growth but did not cause complete tumor regression , 
 and this is in agreement with previous studies . in a study , we evaluated the antitumor efficacy of a synthetic long 
 peptide ( slp ) covering hpv peptide encapsulated in polymeric nanoparticles 
 ( nps ) in a therapeutic tumor model .
similar 
 to the results described here we showed that the peptide vaccine in 
 the particulate form without adjuvant ( poly i : c ) did not significantly 
 delay tumor growth or prolong the survival of mice in comparison with 
 untreated mice .
interestingly , peptide - loaded nanoparticles with poly 
 i : c ( encapsulated or soluble ) significantly prolonged the survival 
 of mice compared to the untreated group . although there are differences 
 between the two studies in the experimental settings , such as the 
 nature of the particulate carrier , type , and dosage of the adjuvants 
 and length of hpv peptide vaccines , similar results in antitumor efficacy 
 for the adjuvanted particulate systems in both studies were observed . 

these findings support our hypothesis that by simply extending the 
 peptide epitopes with a self - assembling segment , a particulate antigen - delivery 
 system can be established without laborious processing and formulation 
 steps .
we conclude 
 that nanoparticle formation based on the introduced 
 self - assembling materials for peptide vaccines can be an attractive 
 approach and simple alternative for other particulate systems .
the 
 extension of minimal epitopes by a self - assembling segment can efficiently 
 augment the induction of a desired immune response : first , by risk 
 reduction of t cell anergy since the segments have to be processed 
 by dcs to release the minimal epitopes ; second , by enhancing cellular 
 uptake of particulate vaccines and antigen - specific t cell response . 

the described approach exploits the advantages of self - assembling 
 peptide epitopes to improve tumor - specific t cell response for cancer 
 treatment . in conclusion
peptide 
 and peptide  polymer conjugates are attractive systems for molecularly 
 well - defined vaccines .
 the aim of the present study was 
 to improve the immunogenicity 
 of peptide epitope vaccines using novel nanocarriers based on self - assembling 
 materials . 
 several studies demonstrated that peptide antigens in nanoparticulate 
 form induce stronger immune responses than their soluble forms . 
 however , 
 several issues such as poor loading and risk of inducing t cell anergy 
 due to premature release of antigenic epitopes have challenged the 
 clinical success of such systems . in the present study 
 , we developed 
 two vaccine delivery systems by appending a self - assembling peptide 
 ( ac - aavvlllw - cooh ) or a thermosensitive polymer poly(n - isopropylacrylamide ( pnipam ) to the n - terminus of different peptide 
 antigens ( ova250264 , hpv - e74357 ) to generate self - assembling peptide epitopes ( sapes ) . 
 the obtained 
 results showed that the sapes were able to form nanostructures with 
 a diameter from 20 to 200 nm . 
 the sapes adjuvanted with cpg induced 
 and expanded antigen - specific cd8 + t cells in mice . 
 furthermore , 
 tumor - bearing mice vaccinated with sapes harboring the hpv e74357 peptide showed a delayed tumor growth and an increased 
 survival compared to sham - treated mice . in conclusion , self - assembling 
 peptide based systems increase the immunogenicity of peptide epitope 
 vaccines and therefore warrants further development toward clinical 
 use . 
Introduction
Experimental
Section
Results and Discussion
Conclusion