recombinant influenza viruses represent a promising delivery system of foreign antigens that can be fused to viral proteins , such as hemagglutinin ( ha ) , neuraminidase ( na ) , or nonstructural protein 1 ( ns1 ) , or can be expressed as an individual whole protein flanked with na segment - specific packaging signals [ 15 ] . influenza viruses bearing model antigens have been used as tools to address several questions and long - standing issues in immune biology . in particular , recombinant influenza viruses bearing the single immunodominant cd8 + t cell epitope ova257264 or the cd4 + t cell epitope ova323339 of chicken ovalbumin ( ova ) have been used in several studies to characterize t cell responses [ 612 ] . ha engineered to carry small polypeptides fused to its n - terminus is efficiently expressed and stably incorporated into retroviral particles without affecting its fusion activity . recently , this strategy was adopted to generate recombinant influenza viruses bearing relatively large domains of the protective antigens of bacillus anthracis toxin ( domain 1 , the region responsible for binding to the lethal factor and the edema factor ( lef ) and domain 4 , the receptor - binding domain ( rbd ) ) fused to the n - terminus of the ha of a / aichi/2/68 ( h3n2 ) in the genetic background of a / wsn/33 virus ( h1n1 ) [ 14 , 15 ] . the chimeric fusion proteins , named ha / lef and ha / rbd , were functionally expressed during virus infection in mice and elicited antibody responses specific to both the viral ha and the protein domains of the bacillus anthracis toxin . here , by using a similar approach , we generated a recombinant influenza virus , wsn - ovai / ii , bearing both ova - specific cd8 + and cd4 + epitopes in its ha , and characterized the ova - specific t cell immune response in vitro and in vivo as well as its ability to protect mice from challenge with the ova - expressing tumor cells b16-ova in a prophylactic model of vaccination . female c57bl/6j mice and the tcr - transgenic mouse line ot - ii , expressing a tcr that recognize the ova323339 epitope , were obtained from charles river , calco , italy . the tcr - transgenic mouse line ot - i , expressing a tcr that recognize an h-2-restricted ova257264 epitope , siinfekl , was kindly supplied by dr . m. bellone ( san raffaele scientific institute , milan , italy ) . for all experiments , mice , aged 612 weeks , were maintained at the istituto superiore di sanit by following institutional guidelines and approved protocols . the coding sequences for the ova257264 ( siinfekl ) and ova323339 ( isqavhaahaeineagr ) t cell epitopes were inserted into the ha gene of a / wsn/33 virus , between the coding regions that correspond to the c - terminus of the ha signal peptide ( sp ) and the n - terminus of the protein following signal peptidase cleavage . specifically , a pair of complementary oligonucleotides encoding the ova epitopes , designated ovai / ii , with flanking clai / psti sites ( encoding esi and lq amino acids , resp . ) was inserted into the cloning cassette generated at the 3 end of the signal peptide coding sequence of the ppoli - wsn ha plasmid , and containing the ggggd peptide arm , as described by li et al . ( figure 1 ) . the recombinant influenza wsn - ovai / ii virus bearing both of the ova - specific cd8 + and cd4 + epitopes was generated by use of plasmid - driven reverse genetics , as described by neumann et al . . the presence of the inserted sequence was confirmed by sequencing . for the antigen presentation assays in vitro , cd8 + and cd4 + t cells were isolated from spleens and lymph nodes of ot - i and ot - ii ova tcr transgenic mice , respectively , by use of magnetic sorting ( miltenyi biotec ) , according to the manufacturer 's protocol . cd11c - positive dendritic cells ( dcs ) were isolated from the spleens of c57bl/6 mice by using macs beads ( miltenyi biotec ) and the purity of the enriched cd11c - positive cell preparation ranged from 90% to 95% . dcs were resuspended to 10  cells / ml in serum - free rpmi 1640 , and incubated with either live or heat - inactivated ( hi ) wsn - ovai / ii virus ( 1000  hau / ml ) for 1  h at 37c , 5% co2 . heat inactivation was performed by incubating wsn - ovai / ii virus at 56c for 30  min in a water bath , and virus fusion activity and inactivation were determined as previously described . after incubation , antigen - loaded dcs were washed and cultured with 10 enriched ot - i or ot - ii cells for 48  h , after which 0.5  ci / well [ h]thymidine ( amersham biosciences , uk ) was added for 16  h before harvesting . for the carboxyfluorescein succinimidyl ester-(cfse- ) based lymphoproliferation assay , live virus - pulsed dcs were coincubated with 2  10 cfse - labelled ot - i or ot - ii cells ; 72  h later , the cells were stained with anti - cd8-phycoerythrin and anti - cd4-phycoerytrin ( pe ) , respectively , for flow cytometry . five - week - old female c57bl/6j mice , anesthetized with avertin given intraperitoneally , were inoculated intranasally ( i.n . ) with 50  l of wsn - ovai / ii virus at different dilutions ( five mice per dilution ) and observed for 21 days to determine the minimal dose lethal to 50% of mice ( mld50 ) . for virological analysis , mice were inoculated with 3  10  pfu of wsn - ovai / ii virus or 5  10  pfu of wsn virus , and virus titres in the lungs and nasal turbinates were determined 3 days and 6 days after infection by use of mdck cells , as described previously . . immunized with 3  10  pfu of wsn - ovai / ii virus or 5  10  pfu of wsn virus . nine days later , five mice from each group were sacrificed to obtain spleens and mediastinal lymph nodes ( mln ) , and epitope - specific t cell responses were determined by using a mouse ifn- enzyme - linked immunospot assay ( elispot ) kit ( pharmingen , bd ) . specifically , a single - cell suspension from lymphoid tissues was cultured with either ova257264 or the viral nucleoprotein ( np366374 ) peptide , whereas enriched cd4 + t cells from the mln were cultured for 48  h with the ova323339 peptide and irradiated ( 2.500  rads ) nave splenocytes . 3  10 b16-ova cells into groups of mice that had been immunized three weeks earlier with wsn or wsn - ovai / ii virus . twelve days after tumor cell injection , eight mice from each group were sacrificed , and lung metastases were enumerated under a dissection microscope . nonimmunized mice served as nave controls . in addition , the frequency of siinfekl - specific ifn--producing t cells was measured by using the elispot assay on spleen - derived lymphocytes . the recombinant influenza wsn - ovai - ii virus bearing both the ova - specific cd8 + and cd4 + epitopes was generated by introducing the coding sequences into the ha gene of a / wsn/33 virus , after the coding region of the ha signal peptide ( sp ) , and the ggggd peptide arm , as described by li et al . ( figure 1 ) . the rescued virus replicated in tissue culture with a titer ranging from 5  10 to 10 plaque - forming units ( pfu)/ml . we first determined whether the ova - specific t cell epitopes of wsn - ovai - ii virus were efficiently presented to antigen - specific t cells . to this end , the capacity of splenic dcs to process and present both live and hi wsn - ovai - ii viruses was assessed by using an in vitro proliferation assay of nave ova - specific cd8 t and cd4 + t cells derived from ot - i and ot - ii tcr - transgenic mice , respectively . live virus - loaded dcs efficiently processed and presented both ova - specific epitopes on mhc class i and ii molecules , whereas there was no response with uninfected dcs ( figure 2(a ) ) . cfse - labelled transgenic t cells were also used to monitor antigen - specific proliferation due to the progressive halving of the dye fluorescence following cell division . wsn - ovai / ii virus - loaded dcs were responsible for vigorous proliferation of ot - i and ot - ii cells within the cd8 + and cd4 + gated populations , respectively , compared to unloaded- or wsn virus - loaded dcs ( figure 2(b ) ) . furthermore , our data indicate that the ova - specific mhc class i - restricted epitope located on the ha of whole hi wsn - ovai / ii virus was efficiently cross - presented by dcs to cd8 + t cells ( figure 2(a ) ) . the binding and fusogenic functions of hi wsn - ovai / ii virus were intact as assessed by standard hemagglutinating and hemolytic assays , respectively , thus confirming the importance of a functional ha to access the cytoplasm of dcs . the higher t cell proliferation of ot - i compared to ot - ii cells induced by dcs loaded with hi wsn - ovai / ii virus , bearing equimolar amounts of both epitopes , correlates with previous data showing that the cell - associated antigen is more efficiently presented to cd8 + than to cd4 + t cells .   to determine the immunogenicity of the wsn - ovai / ii virus in c57bl/6j mice , we first determined its pathogenicity relative to the wild - type wsn virus . none of the inoculated mice died , even those inoculated with doses of wsn - ovai / ii virus as high as 10  pfu , whereas the wild - type wsn virus killed all of the mice at this dose ( data not shown ) . pulmonary viral loads measured 3 and 6 days after infection in mice inoculated with 3  10  pfu of wsn - ovai / ii virus ( average titres of 4.1 and 2.3  log10 tcid50/lung , resp . ) were about four logs lower than those observed for mice inoculated with 5  10  pfu of the wild - type wsn virus ( figure 3 ) . moreover , we could not detect virus in the nasal turbinates of the wsn - ovai / ii - infected mice , whereas detectable viral titres were measured in the nasal turbinates of the wsn - infected animals on day 3 and 6 after infection ( data not shown ) . although the recombinant virus did not replicate as efficiently as the wsn virus , we used the above viral doses to immunize mice and measured comparably high levels of np - specific cd8 + t cells for both viruses in the mln and in the spleen at day 9 after infection ( figure 4(a ) ) . the ova - specific cd8 + t cell response was also vigorous , as determined by ex vivo ifn- release by lymphoid cells after the addition of specific peptides ( figure 4(a ) ) indicating that the attenuated recombinant virus was efficiently immunogenic for cd8 + t cells . to assess whether the engineered ova - specific cd4 + t cell epitope was also presented in the context of infection , we measured the ova323339-specific cd4 + t cells secreting ifn- in the enriched cd4 + t cell population from the mln of these mice . wsn - ovai / ii - infected mice produced an average of 120 spot - forming cells ( sfc)/million cells , whereas wsn - infected mice gave no ova - specific response ( figure 4(b ) ) . although the insertion of foreign epitopes into the ha of wsn - ovai / ii virus was responsible for the attenuated phenotype , the double ova recombinant virus was still replicating and inducing a remarkable ova - specific t cell response in mice . recombinant viral vectors expressing the immunodominant ova - specific cd8 + t cell epitope induce protective immunity against challenge with lethal doses of malignant melanoma cells expressing ova [ 20 , 21 ] . moreover , recombinant influenza viruses expressing a tumor - associated antigen have been previously shown to elicit efficient antitumor responses and to induce tumor inhibition in experimental murine cancer models [ 22 , 23 ] . we , therefore , examined the ability of wsn - ovai / ii virus to protect mice against tumors by using the tumor cell line b16-ova ( ova - transfected b16 cells ) , which cause pulmonary metastases when given intravenously . immunization with wsn - ovai / ii virus protected mice from lung metastases , as measured 12 days after tumor challenge , compared to nonimmunized mice ( figure 5(a ) ) . the antitumor immunity was ova - specific , as all of the mice immunized with the parental wsn virus developed tumors . we also found that wsn - ovai / ii virus - immunized mice produced a stronger ova257264 ( siinfekl-)specific cd8 + t cell response than did either the nonimmunized mice or the wild - type wsn virus - immunized mice , as measured by ex vivo elispot with pooled spleens on day 12 posttumor challenge ( figure 5(b ) ) . the higher ova257264-specific cd8 + t cell response detected in wsn - ovai / ii virus - immunized mice that were subsequently challenged with b16-ova cells correlated with the presence of ova - specific memory t cells in these mice before tumor challenge . here , we engineered a recombinant influenza wsn - ovai / ii virus to bear the ova - specific cd4 + and cd8 + t cell epitopes on its viral ha . the effective proliferation of ova - specific cd8 + t cells in vitro with hi wsn - ovai / ii virus - loaded dcs is in agreement with previous data showing that inactivated influenza viruses that maintain their fusogenic properties are efficiently cross - presented by dcs . the presence of both ova t cell epitopes on the same viral ha protein provides an additional tool , along with ova - specific tcr transgenic mice , to investigate antigen processing and presentation by nonreplicating influenza virus - loaded dcs , in the context of mhc class i and class ii . the double ova recombinant virus thus represents an attractive delivery system for use in studies that explore new vaccination strategies for inducing virus - specific memory cd8 + t cells . moreover , the finding that foreign epitopes located on the ha of inactivated recombinant influenza virions are efficiently cross - presented to specific cd8 + t cells offers interesting opportunities to further explore the use of recombinant influenza virus - based nonreplicating immunogens . in particular , this feature could be used to develop virosomes , as antigen delivery systems , since antigen encapsulation inside the virosomal particles is usually required for cytosolic delivery upon membrane fusion and for proteasome processing to induce ctl responses . our results also show that immunization with wsn - ovai / ii virus protects mice from b16-ova tumor challenge . here , we did not directly address the question of whether the double ova recombinant virus has advantages over the existing recombinant viruses that bear the single ova epitopes . although the induction of ova257264 ( siinfekl-)specific cd8 + t cells in the context of viral infection may be sufficient to provide protective immunity , it is plausible that the simultaneous induction of tumor - specific cd4 + t cells may participate in interactions that shape the protective response against tumors . this is particularly true if tumor - specific immune responses are induced with nonreplicating immunogens and thus in the presence of a poor inflammatory response . recent studies suggest that abnormalities of the immune response in tumor - bearing hosts correlate with the induction of immune tolerance toward tumor - associated antigens and with the functional suppression of antitumor t cell effectors due to myeloid - derived suppressor cells [ 25 , 26 ] . thus , combining wsn - ovai / ii virus infection and adoptive transfer of ova - specific tcr transgenic t cells in mice bearing ova - expressing tumors may help dissect the immunoregulatory mechanisms responsible for tumor - induced tolerance and immune suppression and may provide useful information for rational design of cancer immunotherapy .
recombinant influenza viruses that bear the single immunodominant cd8 + t cell epitope ova257264 or the cd4 + t cell epitope ova323339 of the model antigen ovalbumin ( ova ) have been useful tools in immunology . here , we generated a recombinant influenza virus , wsn - ovai / ii , that bears both ova - specific cd8 + and cd4 + epitopes on its hemagglutinin molecule . 
 live and heat - inactivated wsn - ovai / ii viruses were efficiently presented by dendritic cells in vitro to ot - i tcr transgenic cd8 + t cells and ot - ii tcr transgenic cd4 + t cells . in vivo , wsn - ovai 
 / ii virus was attenuated in virulence , highly immunogenic , and protected mice from b16-ova tumor challenge in a prophylactic model of vaccination . 
 thus , wsn - ovai / ii virus represents an additional tool , along with ova tcr transgenic mice , for further studies on t cell responses and may be of value in vaccine design .