Recombinant Adeno-Associated Virus Encoding Epstein-Barr Virus Latent Membrane Proteins Fused With Heat Shock Protein DNA As A Potential Vaccine For Nasopharyngeal Carcinoma

Background and objective:Nasopharyngeal carcinoma (NPC) is a common cancerin Southern China and Epstein-Barr virus (EBV) is the most important pathogenesis.Hence,there is considerable interest in the possibility of targeting the virus-specific immuneresponse to the viral antigens,which are expressed in these malignancies.These antigensinclude LMP1,and LMP2.Preview studies have shown a low CTL precursor frequency toepitopes within LMP1 in healthy virus carriers,suggesting that reconstitution of bothLMP1- and LMP2- specific CTL responses may be necessary for a long-term therapeuticbenefit of NPC patients.Adeno-associated virus (AAV),a single-stranded virus,has beenstudied as a vector for gene therapy.AAV has many natural features that are attractive for ahuman gene therapy vector,such as nonpathogenicity,targeted integrating capability,and abroad host range.In this study,we explore a potential that recombinant adeno-associatedvirus (rAAV) carrying a fusing gene containing heat shock protein as an adjuvant,EBVlatent membrane proteins cytotoxic T-lymphocyte epitope DNA as a vaccine preventsNasopharyngeal carcinoma.The tumor vaccine was devised by constructing a chimericgene which contained EBV latent membrane proteins cytotoxic T-lymphocyte epitopeDNA fused with the heat shock protein gene as a tumor vaccine delivered via rAAV.Method:1.First we cloned the coding sequence of EBV LMP1 and LMP2,andconstructed pAAV-LMP2/1-hsp.The rAAV-LMP2/1-hsp was obtained by calciumphosphate-DNA co-transfection methods with the help of packaging-plasmid pXX2 andhelper-plasmid phelper.The control rAAV-GFP was simultaneously packed using the samemethods,followed by purification and tittering of the purified rAAVs with real-time PCRtechniques.2.The SP2/0 cell line was transfected with plasmid pCDNA3.1-LMP2 to allow stable LMP2 expression,and the positive cell clones were selected bv G418 (400ug/ml) andnamed SP 2/0-LMP2 cell line.3.LMP2_228-242 synthetic peptide was injected beneath theskin of two New Zealand white rabbits.After 6 weeks,rabbits were sacrificed,and theirblood was collected and purified to acquire rabbit Anti-LMP2 polyclonal antibodies.4.Todetermine whether the chimerically constructed LMP2/1CTL-hsp gene can be expressedvia rAAV delivered to cells,we infected 293 cells using rAAV-LMP2/1-hsp.Two dayslater,the cellular protein was harvested and the expression of the LMP2/1-hsp chimericgene was analyzed by Western blotting with rabbit Anti-LMP2 polyclonal antibodies.5.Two different vaccination strategies were used to assess the efficacy of the rAAV vaccine.In the first set of experiments,BALB/c mice were immunized with eitherrAAV-LMP2/1-hsp or rAAV GFP.Three weeks after the immunization,these mice werechallenged s.c.with live SP2/0-LMP2 or SP2/0 cells.After challenge,these animals wereregularly monitored for 30 days,and the tumor size was measured by calipers.In thesecond set of experiments,BALB/c mice were first challenged with SP2/0-LMP2 or SP2/0tumor cells.Ten days after the challenge,mice were immunized i.m.with either rAAVLMP2/1-hsp or rAAV GFP.The therapeutic efficacy of the rAAV vaccine was assessed byregular monitoring of tumor regression.6.To assess the Cellular immune response of therAAV vaccine,BALB/c mice were vaccinated with recombinant virus encoding LMP2/1,rAAV GFP,or normal saline.After 3 weeks,mice were sacrificed,and Spleen cells wereretained.Target cells were SP2/0-LMP2 or WT SP2/0,and Effector cells were T cells.LDH release was measured using the Cytotox 96 cytotoxicity assay.7.To analyse theimmune mechanism of the rAAV vaccine,BALB/c mice were vaccinated withrrAAV-LMP2/1-hsp,rAAV-GFP,or normal saline.After 3 weeks,mice were sacrificed,and T cells were pulsed in vitro with or without LMP2 synthetic peptide (PYLFWLAAI),or pulsed with nonspecific peptide for a 4-day proliferation assay.Proliferation wasquantified by MTT Assays.Results:1.Restriction enzyme analysis,PCR and sequencing results showed that therecombinant plasmid pAAV-LMP2/1-hsp was successfully constructed.2.Western blotsshowed that recombinant protein was detected by anti-LMP2 antibodies in rAAV-LMP2/1-hsp infected cells but not in the control cells infected with rAAV-GFP.3.BALB/c mice (6 mice in each group) were first immunized with rAAV LMP2/1-hsp orrAAV GFP,and then challenged with SP2/0 or SP2/0-LMP2 cells.Although both groups ofanimals developed tumors,the tumor outgrowth in rAAV-GFP was highly aggressive.Inthe contrast,these tumors grew much less aggressively in animals immunized withrAAV-LMP2/1-hsp,and outgrowth was completely resolved in 90% of the animals by theend of the observation period in the mice with load of SP2/0-LMP2 cells.By day 35,theaverage tumor load in rAAV LMP2/1-hsp immunized mice was 5 fold lower whencompared with rAAV GFP immunized mice.4.In the second set of experiments,followingimmunization,the tumor size progressively increased in almost all animals injected withrAAV-GFP,and by day 20 after immunization,100% mice were dead.In contrast,adramatic reduction in the tumor load was observed in mice immunized with rAAVLMP2/1-hsp,and showed long-term protection.5.T cells from BALB/c mice immunizedwith rAAV-LMP2/1-hsp,rAAV-GFP,or mock were isolated and stimulated in vitro withLMP2 synthetic peptide.The results showed that SP2/0-LMP2 and WT SP2/0 cells pulsedwith LMP2 peptide 131 to 139 were lysed,whereas WT SP2/0 cells alone or WT SP2/0cells pulsed with nonspecific peptide were not lysed.6.T cell proliferation assay showedthat T cells from mice vaccinated with rAAV-LMP2/1-hsp could be stimulated mice byLMP2 peptide_131-139.Conclusions:we successfully developed rAAV encoding EBV LMP2/1 CTL peptideDNA fused with hsp DNA as a tumor vaccine,which may induce CTL response to retardtumor growth.It is a potential vaccine for nasopharyngeal carcinoma treatment using hsp asa carrier protein and delivery by rAAV vector.