Preparation Of HLA-Specific HCMV Polyepitope Ad5F35 Vectored Nucleotide Vaccine In Chinese People And Valuation Its Immune Activity

BackgroundHuman cytomegalovirus(HCMV) is the most complex of the eight human herpesvirus species and has a 235-kb double-stranded DNA that encodes 165 genes.HCMV infects approximately 40-60%of the developed world's population.Primary infection in healthy hosts is usually asymptomatic,and the virus persists in CD33 progenitors expressing markers of dendritic and myeloid lineage as major reservoirs of latent infection virus without any apparent clinical symptoms.HCMV-associated clinical disease has been recognized in three populations:Neonates with immature immune systems,transplant recipients with impaired immune systems due to the use of drugs that suppress rejection and HIV-infected patients with impaired immune systems due to the decline of CD4+T cellsHCMV is the most important viral pathogen affecting transplant recipients,including both solid organ transplant and allogeneic hematopoietic stem cell transplant recipients whose immune systems are suppressed by drugs used to prevent graft rejection.It is noted that HCMV is reported to infect not only stroma and epithelial cells but also hematopoietic cells including CD34+ stem cells,monocytes,and dendritic cells. Late-onset cytomegalovirus disease in hematopoietic stem cell transplant recipients(later than day 100 after transplantation) remains a major cause of morbidity and mortality, despite the introduction of new antiviral agents.Congenital HCMV infection is the most common intrauterine infection and 0.3-2.4%of neonates are born with HCMV infection worldwide.Of the neonates with congenital HCMV infection,10%have symptoms of irreversible CNS involvement in the form of microcephaly,encephalitis,seizures, deafness,upper-motor neuron disorders and psychomotor retardation.Intensive study over the last 30 years has demonstrated that both innate and adaptive immunity play important roles in controlling latent HCMV infection.The innate immune response,mainly natural killer cells,may shape or augment the adaptive immune response against HCMV infection.NK cells may act by secreting IFN-γ,which can facilitate the expansion of antigen-specific helper T cells to control HCMV infection.The adaptive immunity,including both humoral and cellular immunity,plays a crucial role in controlling HCMV infection.Extensive analysis of humoral immune responses to HCMV has revealed that response is directed toward multiple viral protein and that virus-protection antibodies are directed primarily to the envelope glycoprotein of HCMV with glycoprotein B(gB) as a dominant target antigen.HCMV-specific T-cell immunity is the most important adaptive immune component responsible for suppressing HCMV and keeping it at latency after primary infection.Studies showed that pp65 and IE-1 constituted 40%of the total CD8+ T-cell responses and 60%of these T-cell responses are directed towards other antigens,such as pp28,pp150,pp71 and US proteins.Compared with the CD8+ T cells,CD4+ T cells were generally thought to have an indirect role in controlling HCMV infection by providing helper signals for the generation of antibodies and the maintance of CD8+ T-cell memory.Taken together,our increasing understanding of immune responses against HCMV infection has firmly established that activation of both humoral and cellular immunity is crucial for successful HCMV vaccine.Partâ… Prediction and Decision of HLA-specific HCMV Polyepitope in Chinese PeopleObjective:Prediction and decision of HLA-specific HCMV polyepitope in Chinese people.Methods:1.According to the forecasting system on spatial coverage of cumulative phenotypic frequency(CPF) of HLA-I of professor XUE Fu-zhong in Institute of Epidemiology and Medical Statistics of Medical College of Shandong University,the study forcasted the CPF of 14 HLA allel sites including A2,A24,A1,A3,A11,A68,B44,B7,A23,A26, B35,B38,B8,B27 in Chinese people and evaluated immune activity of HLA-specific HCMV polyepitope nucleotide vaccine in different geographical location of Chinese people.2.Utilizating 23 SCI literature about HCMV epitope which have been published in PubMed since 1996,the study selected multiple CTL epitopes,Th epitopes and B cell epitopes in different HCMV protein antigens which can induce cellullar immunologic response and humoral immunoresponse and stably bind classâ… and classâ…¡MHC molecules.3.Through SYFPEITHY combined with MAPPP algorisms,the selected epitopes were predicted for the specific value of peptide and proteasomal cleavage site.4.The nucleotide sequences of the selected epitopes were found in genome of HCMV AD169 strain and verified in PubMed Nucleotide NC₀01347.Furthermore,the nucleotide sequences of these epitope were cascaded a single nucleotide sequence according to frequency distribution of HLA allel.5.According to Kozak rule,Kozak sequence was put into the nucleotide sequence of HCMV polyepitope which can induce highly efficient expression in eukaryotic expression vector.6.The nucleotide sequence of HCMV polyepitope was input DNAMAN software. Enzyme cleavage sites of the nucleotide sequence were decided in order to construct shuttle vector in the next experiment.Results:1.The coverage of HLA-specific HCMV polyepitope nucleotide vaccine in Chinese people has reached above 90%.2.The epitopes included in this polyepitope were derived from 15 different viral proteins(pp28,pp50,pp65,pp150,pp71,gH,gB,IE-1,IE-2,US2,US3,US6,US11, UL16 and UL18) involved in virus attachment,replication,assembly,reactivation and immune escape from the latent phase,all of which are crucial stages in the development of HCMV disease.There were 76 CTL epitopes,7 Th epitopes and 1 B cell epitope in selected epitopes.In theory,these epitopes can induce cellular immunologic response and humoral immunoresponse and prevent the development of HCMV disease.3.Through the SYFPEITHI web site,the predicted values of the selected epitopes have reached above 14 scores,almost between the 20 and 30 scores.Through the MAPPP server,the predicted proteasomal cleavage values of the selected epitope have reached above 0.5 score.4.After introducing Kozak rule,adding 6×his identify tag and utilizating genome of HCMV AD 169 strain in PubMed Nucleotide NC₀01347,the nucleotide sequence of the polyepitope has been decided.5.In order to construct shuttle vector in the next experiment,the Nheâ… and Notâ… enzyme cleavage site,of the nucleotide sequence have been decided after the nucleotide sequence of HCMV polyepitope was input DNAMAN software.Conclusions:1.According to the forecasting system on spatial coverage of cumulative phenotypic frequency(CPF) of HLA-â… ,the CPF of 14 HLA allel sites including A2,A24,A1,A3, A11,A65,B44,B7,A23,A26,B35,B35,B8 and B27 in Chinese people has reached 92.1%.It has exhibited the coverage of HLA-specific HCMV polyepitope nucleotide vaccine in Chinese people has reached above 90%.It also has evaluated immune effect of HLA-specific HCMV polyepitope nucleotide vaccine in different geographical location of Chinese people.In theory,immune effect of HLA-specific HCMV polyepitope nucleotide vaccine can reach above 90%.2.The epitopes included in this polyepitope were derived from 15 different viral proteins(pp28,pp50,pp65,pp150,pp71,gH,gB,IE-1,IE-2,US2,US3,US6,US11, UL16 and UL18) involved in virus attachment,replication,assembly,reactivation and immune escape from the latent phase,all of which are crucial stages in the development of HCMV disease.There were 76 CTL epitopes,7 Th epitopes and 1 B cell epitope in selected epitopes.In theory,these epitope can induce cellular immunologic response and humoral immunoresponse and prevent the development of HCMV disease.3.After introducing Kozak rule,utilizating genome of HCMV AD169 strain in PubMed Nucleotide NC₀01347 and inputting DNAMAN software,the nucleotide sequence of the polyepitope has been decided.Kozak sequence has been added in the upstream that can induce high efficient expression in eukaryotic expression vector.Six×His identify tag has been added in the downstream that can be used in Western blot and cell immunochemistry experiment.The output Nheâ… and Notâ… enzyme cleavage site can be used to construct shuttle vector in the next experiment. Partâ…¡Preparation of Novel Vaccine against HCMV Based on Ad5F35 Adenovirus Vector Expressing HLA-specific polyepitope nucleotideObjective:HCMV disease accounted for the high morbidity and mortality in transplant patients and new born babies.The worldwide institute of medicine has assigned the highest priority for a vaccine to control HCMV disease.In spite of numerous attempts, successful licensure of a HCMV vaccine formulation remains elusive.The modified replication-deficient adenoviral vector AdSF35 possesses more expanded tropism and larger foreign DNA package ability than the conventional Ad5 vectors without affecting the viral growth rate and titer.It exhibits high transduction efficiency in many human cells.Here we have developed two novel chimeric vaccine strategy based on AdSF35 which encode multiple HLA classâ… &â…¡restricted CTL and Th epitopes from HCMV as a contiguous polypeptide and encode the high conserved predominant B cell epitope gB AD-1.Methods:1.Using SOEing and PCR technology to synthesize the total length CTL·Th gene of HCMV multiple T cell epitope.2.The AD-1 gene was amplified from AD169 genomic DNA by polymerase chain reaction(PCR) with the oligonucleotide primer.3.The CTL·Th gene and AD-1 amplicon were ligated the Nheâ… /Notâ… and Kpnâ… /Aflâ…¡sites of shuttle plasmid pHMCMV5 respectively.The desired plasmids were identified by restriction analysis and nucleotide sequence analysis.4.The expression cassette,comprising the CMV promoter,CTL·Th gene/AD-1 and an SV40 polyA signal,was excised from the shuttle plasmids pHMCMV5-CTL·Th or pHMCMV5-AD-1 with I-CeuI and PI-SceI and ligated into adenoviral backbone vector pAd5F35 cleaved with the same enzymes.The presence of expression cassettes was verified by digestion with Xhoâ… andâ… -Ceuâ… /PI-Sceâ… . 5.pAd5F35-CTL·Th and pAd5F35-AD-1 were linearized with PacI and transfected into HEK293 cells using lipofectamine^TM 2000.Transfected HEK293 cells (HEK293-Ad5F35-CTL·Th or HEK293-Ad5F35-AD-1) were incubated at 37℃for 7-10 days until maximal virus cytopathic effect(CPE) was observed.Then recombinant adenovirus was harvested.6.The recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 were amplified using HEK293 cells until the 5^th passage.The titer of the large-scale recombinant adenovirus was established by plaque assay according to adenovirus TCID50 testing kit protocol.7.The expression of CTL·Th and AD-1 was identified by using transfected HEK293 cells.To confirm CTL·Th and AD-1 mRNA expression,RT-PCR analysis were conducted.To confirm AD-1 protein expression,western blot analysis was conducted.8.The expression of CTL·Th and AD-1 in PBMCs infected with Ad5F35-CTL·Th and Ad5F35-AD-1 was analyzed by immunocytochemistry,and cell viability were determined by trypan blue staining.Results:1.Using SOEing and PCR technology,the total-length gene of HCMV multiple T cell epitopes has been successful synthesized.2.AD-lgene has been successfully amplified from the genome of HCMV AD169 strain.3.Shuttle plasmids pHMCMV5-CTL·Th and pHMCMV5-AD-1 have been successfully constructed.After enzyme digestion identification and DNA sequencing, insertion of the gene was proved correct.4.Recombinant adenovirus vectors pAd5F35-CTL·Th and pAd5F35-AD-1 have been successfully constructed.After enzyme digestion identification and DNA sequencing,the expression of the gene was proved correct.5.The recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 have been successfully packaged.Full CPE was observed for recombinant virus after transfecting HEK293.6.The recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 were amplified using HEK293 cells until the 5^th passage.Each cycle of amplification only needed 2-3 days.7.The titer of the large-scale recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 virus preparation were 2.5×10⁹ PFU by plaque assay.8.After transfecting HEK293 cells using recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1,CTL·Th and AD-1 expression were verified at the level of mRNA protein.9.After PBMCs were transfected 10 day using Ad5F35-CTL·Th and Ad5F35-AD-1, 90%of cells retained viable.It has shown no any adverse effects of the recombinant adenovirus.10.Strong expression of CTL·Th polyepitope antigen and AD-1 were recorded in PBMCs infected with Ad5F35-CTL·Th and Ad5F35-AD-1.The typical pattern of staining demonstrates that recombinant CTL·Th polyepitope localizes to the cell membrane and cytoplasm,while the peripheral pattern of staining demonstrates that recombinant AD-1 localizes to the cell membrane.Conclusion:1.Two novel vaccines based on a replication-deficient adenovirus that encode HLA-specific HCMV polyepitope nucleotide vaccine(referred to as Ad5F35-CTL·Th) and a single B cell epitope gB AD-1(referred to as Ad5F35-AD-1) were successfully constructed.2.Ex vivo stimulation of PBMC with Ad5F35-CTL·Th and Ad5F35-AD-1 consistently showed highly efficient expression of CTL·Th polyepitope antigen and epitope AD-1 protein.The viability of PBMC was not affected.It will be useful for HCMV novel vaccine development.Partâ…¢Immune Activity Evaluation of Novel Vaccine against HCMV Based on Ad5F35 Adenovirus Vector Expressing HLA-specific polyepitope nucleotide Objective:The adaptive immunity,including both humoral and cellular immunity, plays a crucial role in controlling HCMV infection.This experiment is to evaluate immune activity of this novel vaccine against HCMV based on a chimeric Ad5F35 adenovirus vector expressing HLA-specific polyepitope nucleotide.Methods:1.Lymphoblastoid cell lines-LCL is established as antigen presenting cell by using EBV to transform PBMC.2.In vitro antigen-specific CTL was generated from a panel of healthy virus carriers by stimulating PBMC with adenoviral chimeric vaccine Ad5F35-CTL·Th.3.These CTL clone lines were screened for cytotoxic activity on a panel of autologous target cells that were either sensitized with synthetic peptides or infected with Ad5F35-CTL·Th.4.The ELISPOT assay was used to assess whether adenoviral chimeric vaccine Ad5F35-CTL·Th could stimulate a memory response,as measured by the production of IFN-γ,in PBMC from a large panel of seropositive donors.5.The Intracellular cytokine staining(ICS) assay was used to measure the levels of specific CD8+T cells elicited by Ad5F35-CTL·Th stimulating PBMC from healthy seropositive individuals.Results:1.Two EBV-transformed-LCLs have been successfully established.2.In vitro stimulation with this adenoviral chimeric vaccine Ad5F35-CTL·Th rapidly expanded multiple antigen-specific human CD8+T-cells from healthy virus carriers.3.The HCMV epitopes encoded by this polyepitope were endogenously expressed and processed by human cells.4.The production of IFN-γhas been largely secreted from PBMC stimulated by Ad5F35-CTL·Th or expanded antigen-specific CTL.5.The levels of specific CD8+T cells have been strongly elicited by Ad5F35-CTL·Th stimulating PBMC from healthy seropositive individuals.Conclusion:1.This study shows the effectiveness of a polyepitope antigen presentation system for reproducible expansion of antigen-specific T cells from immunocompetent settings.2.It also shows that an adenovirus based polyepitope is highly efficient in expanding both immunodominant and subdominant antigen-specific T cells.3.The comparable expansion of both immunodominant and subdominant T cell responses using a single expression construct was an unexpected result,as previous studies have shown that the different T cell populations require distinct stimulation strategies.4.The data presented here emphasize the strength and adaptability of the polyepitope-based approach for clinical translation and may lead to significant advances in the application of adoptive immunotherapy to a wide range of diseases.It will be useful for HCMV novel vaccine development.