hepatocellular carcinoma ( hcc ) is one of the most common malignancies in the world ( 700 , 000  deaths / year ) and its incidence is increasing . therapeutic options available include liver transplantation , surgical resection and local ablative therapies , but the prognosis of hcc remains poor due to high tumor recurrence rates . thus , new strategies are needed and immunotherapy based on stimulation of the antitumor immune response is a promising approach . . secreted afp , synthesized in the yolk sac , the fetal liver , and the gastrointestinal tract , is the main serum protein during the fetal development . however , afp is reexpressed in 50 to 80% of hcc , whereas its expression is low or absent in normal adult liver . several vaccination strategies based on afp as a taa have been investigated for hcc immunotherapy . briefly , afp can be presented as an antigen by human and murine dc and persisting t cd8 memory clones present in the human and the murine repertories can recognize some peptide epitopes derived from afp [ 58 ] . in murine models , murine afp can function as a tumor rejection antigen in melanoma models   and afp - specific vaccination can significantly impair the growth of an autochthonous hepatocellular carcinoma . in the two conducted clinical trials , afp - specific ctl have been detected in blood after afp peptides administration but , however , it has not been correlated to an objective clinical response [ 7 , 8 ] . this could be due to the fact that onco - fetal antigens , including afp , are self - tolerated molecules and that the tumor immune evasion mechanisms have to be successfully controlled to trigger an efficient antitumor response . vaccination with altered forms of the antigen or with the xenogeneic protein is a possible strategy to overcome the tolerance to antigens [ 1113 ] . it has been successfully used to raise immune response against various antigens [ 15 , 16 ] . compared to immunotherapy strategies with monoclonal antibodies or with immunodominant peptides , vaccination with mva encoding a tumor antigen presents the advantage of raising an immune response against the whole antigen . in the context of a mva vaccination , the primary induction of the ctl response depends mostly on cross - presentation suggesting that the expression of a full - length antigen localized subcellularly should be considered during the mva vector design . in the present study , we compare the immune response elicited by the vaccination with a recombinant mva vector encoding the afp antigen either in its native secreted form or in modified membrane - associated or intracellular forms . primary chicken embryo fibroblasts ( cef ) were used for homologous recombination and amplification of recombinant vectors . cef were prepared from chicken embryos obtained from fertilized eggs ( lohmann , france ) previously incubated 11 days at 37c in a humid atmosphere . embryos were dissected and treated with a triple select solution ( invitrogen ) ( w / v ) . cef cells were maintained in eagle - based medium supplemented with 5% fetal bovine serum . the mva shuttle plasmid contains a multiple - cloning site under the control of the ph5r early - late vaccinia virus promoter surrounded by the flanking sequences of the deletion iii of the mva allowing homologous recombination and a selection gene , the e. coli xanthine - guanine phosphoribosyl - transferase gene ( gpt gene ) under the control of the p11k7.5 promoter . full - length cdna encoding the secreted form of murine afp ( s - mafp ) was obtained by pcr ( primers f5-ggccgcgctagccgccaccatgaagtggatcacacccgcttc-3 ; r5-gcagtcagggcccctgcactcagtaatacataaacgcccaaagcatcacgagttttg-3 ) and digestion with nhei and noti enzymes ( new england biolabs ) on pcineomafp . the intracellular afp sequence ( i - mafp ) was obtained by deleting the exporting signal ( nucleotides 1 to 54 ) by pcr ( primers f5-ggccgcgctagccgccaccatgaaagcattgcacgaaaatgagtt-3 ; r5-gagccacatccagggccagcttc-3 ) and enzyme digestion . the transmembrane afp ( t - mafp ) sequence was obtained by successive pcr and fusion between the s - mafp sequence and a rabies transmembrane domain , kindly provided by dr . the resulting sequences , verified by sequencing , are described in figure 1(a ) and were cloned into the corresponding sites of the mva shuttle plasmid . briefly , cef cells were infected with a mva without any inserted transgene ( mva - null ) and then transfected with the different shuttle plasmids by cacl2 precipitation . homologous recombination occurred and recombinant mva viruses are isolated by multiple steps of mycophenolic acid selection . final recombinant mva viruses were controlled by pcr , amplified in cef , and virus stocks were titrated on cef by plaque assay . cef cells were infected with each mva vector at a multiplicity of infection ( moi ) of 0.2 and incubated for 48  h. cell lysate proteins were run on a 10% sds - page gel under reducing conditions and transferred onto a nitrocellulose membrane . the membrane was incubated with a polyclonal goat anti - afp ( c-19 ) antibody ( santa cruz ) at a 1  :  1000 dilution or with a rabbit anti - rabies transmembrane domain at a 1  :  2000 dilution . the membrane was then washed and incubated with a secondary antibody coupled with horseradish peroxidase ( ge healthcare ) . c3h / hen ( c3h ) male mice ( 8 weeks ) were purchased from janvier laboratory ( france ) and were bred in the nantes ifr 26 animal facility . all experiments procedures involving animals were conducted according to the guidelines of the french agriculture ministry and were approved by the local ethical committee . mice were immunized three times subcutaneously ( s.c . ) into the flank with 5  10  pfu of mva vectors at 7-day intervals . mice subjected to regulatory t cells depletion were injected intraperitoneally ( i.p . ) 1 day prior the vaccination course with 400  g of anti - cd25 antibody ( pc61 ) purchased from bioxcell ( new hampshire , usa ) . cd8 t cells were isolated from spleen cells with mouse cd8a ( ly-2 ) microbeads and ms columns ( miltenyi biotec ) . specific t - cell response was monitored on cd8 t cells with ifn elispot kit ( diaclone ) , according to the manufacturer 's protocol . cells , plated at a concentration of 5  10 cells per well , were stimulated with 20  m of a mafp - specific cd8 restricted peptide ( nefgiastl ) described earlier , with medium alone or with mva - null at moi 10 for 24  h. ifn-secreting cells were counted on an aid elispot reader 9 ( autoimmun diagnostika ) . results are presented as the number of ifn-secreting cells per million of cd8 t cells after subtraction of nonspecific signal obtained with medium alone . blood samples were collected on days 0 , 7 , 14 , and 21 during the vaccination course . plasma was separated by centrifugation from blood collected . a fusion protein consisting of the native murine afp and a v5 tag afp open reading frame was inserted in frame with a sequence encoding the v5 epitope in the pcdna3.1/v5-his vector ( invitrogen ) . 293 cells were transfected with the resulting plasmid with lipofectamine ( invitrogen ) and the afp - v5 fusion protein was secreted in the culture supernatant . 96-well plates were coated overnight at 4c with a goat anti - v5 tag antibody ( abcam , uk ) at 1  g / ml diluted in bicarbonate coating buffer . following blocking with pbs - tween - milk ( 5% ) ( ptm ) , plates were incubated overnight at 4c with 100  l of supernatant containing the afp - v5 fusion protein . wells were then washed three times in ptm and incubated overnight at 4c with primary mouse sera diluted serially in ptm . wells were then washed three times in ptm and incubated with a goat anti - mouse igg hrp secondary antibody ( dako ) for 2  h at room temperature . following washings in ptm , wells were incubated with an abts substrate ( roche diagnostics , germany ) . statistical analyses were performed using the non - parametric kruskal - wallis or mann - whitney tests and graphpad prism software . a p value < 0.05 was considered to be statistically significant . the sequences corresponding to the secreted ( s mafp ) , intracellular ( i mafp ) , and transmembrane ( t mafp ) forms of the murine afp protein were introduced in the deletion iii of the mva genome under the control of the early - late promoter ph5r . the recombinants mva produced are presented in figure 1(a ) . specific signals corresponding to the intracellular and to the secreted mafp proteins in cellular extracts isolated from cep cells infected either by mva - i mafp ( figure 1(b ) , lane 1 ) or by mva - s mafp ( figure 1(b ) , lane 2 ) were detecting using a polyclonal antibody directed against the c - terminal part of the afp protein . the respective sizes of these two proteins are in line with the expected size of the native mafp protein and with the molecular modifications achieved . as the antibody previously used was directed against the c - terminal part of the protein where the fusion was made , the expression of the transmembrane form was confirmed by western blot using an antibody directed against the transmembrane rabies domain . a specific protein band was detected at a higher size compared to the native protein in agreement to the fusion with the supplementary domain ( figure 1(c ) , lane 1 ) . mice ( n = 4 ) were vaccinated with the different recombinants mva by three subcutaneous injections one week apart ( figure 2(b ) ) . one week after the last immunization , the specific cd8 t - cell response was measured in an interferon gamma elispot assay , with the h-2 afp peptide nefgiastl , as described previously . as shown in figure 2(b ) and as expected , no specific response was detected in mice vaccinated with the parental vector mva - null . surprisingly , no significant increase in the number of spots was observed in mice vaccinated either with mva - s mafp or with mva - i mafp . in contrast , mva - t mafp - vaccinated mice displayed a significantly higher number of ifn--producing cells compared to mice vaccinated with the other mva vectors . thus , in the context of afp vaccination by an mva vector , the transmembrane form elicits a higher cd8 t - cell immune response in c3h / hen - vaccinated mice . populations of regulatory t cells could impair the immune response against a self - antigen such as afp . to address this possibility , groups of mice were vaccinated with the mva - t mafp vector , with or without an injection of 400  g of pc61 anti - cd25 monoclonal antibody the day before the first vaccination ( figure 3(a ) ) . at sacrifice , cd4 cd25 t - cell depletion was verified by facs analysis ( data not shown ) and the cd8 t - cell response was compared in splenocytes prepared from the two groups by ifng elispot assay . a single injection of anti - cd25 antibody did not enhance significantly the afp - specific cd8 t - cell immune response ( figure 3(b ) ) . in contrast , the t reg depletion has an impact on the immune response against the vector ( figure 3(c ) ) . in fact , the antibody injection resulted in a significantly higher number of ifn--producing cd8 t cells after mva restimulation confirming that the depletion did occur . to monitor the afp - specific humoral response elicited by the vaccinations with the mva recombinant vectors , mice ( n = 3 ) were vaccinated either with the mva afp viruses or with the mva - null , by three injections one week apart and sacrificed one week after . the serological response was assessed by elisa as described in the material and methods section . the presence at sacrifice of circulating antibodies directed against the afp protein was analyzed in mice vaccinated with the four different mvas ( figure 4(b ) ) . no afp - specific antibody could be detected in the sera of mice vaccinated with mva - null , or mva - s mafp or mva - i mafp , even at the minimal dilution ( 1  :  50 ) . in contrast , vaccination with mva - t mafp elicited a significantly stronger specific antibody response . the time course of afp - specific antibody titer was assessed during the vaccination protocol ( figure 4(c ) ) . repeated immunizations ( at day 0 , day 7 , and day 14 ) with mva - t mafp led to the production of afp - specific antibodies which was maximal by day 14 and remained stable at day 21 . tolerance to tumor - associated antigen is profound , therefore strenuous immune modulation is required to overcome it and achieve meaningful response . in this paper , we assessed the ability of mva to induce an immune response against the self - antigen -fetoprotein ( afp ) , and we compared the immunogenicity of different forms of this antigen after vaccination in nave mice . three vaccines were generated to encode recombinant afp that was either secreted or membrane - bound or cytosolic . comparable strategies targeting antigen to specific subcellular compartments have been shown to efficiently modulate the specific immune response elicited . the correct expression of the recombinant afp forms by the mva vectors was first confirmed . the vaccination protocol consisted in three subcutaneous injections one week apart and one week later , both the cellular - specific cd8 t - cell response and the humoral response were monitored . expression of a cytosolic form of afp failed to raise a strong immune response as what has been shown for a soluble cytosolic fragment of the her-2 protein which is promptly degraded by the proteasome . vaccination with the transmembrane form of afp induced a stronger cd8 t - cell response compared to the ones obtained with the mva encoding the secreted and the intracellular forms of afp . since the number of specific spots was limited , we evaluated the effect of a t reg depletion on the immune response obtained and we concluded that the afp - specific cd8 response was not modulated by the cd25 t cell population during a mva - t mafp vaccination protocol . finally , the vaccination with the transmembrane form of the antigen leads to the production of antibodies directed against afp . previously , vaccination with an mva encoding a modified membrane - associated form of the epithelial tumor antigen h23 , aberrantly expressed in breast cancer , has been shown to allow the production of elevated levels of specific antibodies in vaccinated animals [ 23 , 24 ] . the difficulty to raise a significant immune response against afp compared to other taa could be due to the fact that this self - antigen is produced by the liver , which is known to constitute a specific tolerogenic environment . in conclusion , we demonstrate that the secreted and the intracellular afp antigens expressed from mva are less effective immunogens than their transmembrane counterpart . vaccination with a membrane - bound form of a self - antigen , combined with other strategies , involving stimulation with cytokines ( il2 , il12 , gm - csf ) [ 26 , 27 ] could represent a promising strategy to enhance immunogenicity in the context of mva vaccination .
aim . to investigate the ability of recombinant modified vaccinia virus ankara ( rmva ) vector to induce an immune response against a well - tolerated self - antigen 
 . methods . 
 rmva vectors expressing different form of -fetoprotein ( afp ) were produced and characterized . 
 nave mice were vaccinated with mva vectors expressing the afp antigen in either a secreted , or a membrane - bound , or an intracellular form . 
 the immune response was monitored by an ifn elispot assay and antibody detection . 
 results . 
 vaccination with the membrane - associated form of afp induced a stronger cd8 + t - cell response compared to the ones obtained with the mva encoding the secreted or the intracellular forms of afp . 
 moreover , the vaccination with the membrane - bound afp elicited the production of afp - specific antibodies . conclusions . 
 the afp transmembrane form is more immunogenic . 
 expressing a membrane - bound form in the context of an mva vaccination could enhance the immunogenicity of a self - antigen .