Immunogenicity of DNA and Recombinant Sendai Virus Vaccines Expressing the HIV-1 gag Gene

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.

Two bicistronic DNA vaccines against Vibrio anguillarum and the immune eff ects on flounder Paralichthys olivaceus

Chemokines are cytokines that can promote the activation and migration of immune cells,and increase the recognition of antigen by antigen-presenting cells(APC).Previous studies showed that a DNA vaccine can induce humoral and cellular immune responses of flounder after immunization.To explore the improvement of chemokines on the efficiency of OmpK vaccine,two bicistronic DNA candidate vaccines were constructed and the immune responses they induced in the flounder were investigated by reverse transcription polymerase chain reaction(RT-PCR),indirect immunofl uorescent assay(IFA),H&E staining,fl ow cytometry(FCM),and quantifi cational real-time polymerase chain reaction(qRT-PCR).pBudCE4.1 plasmid as an expression vector,bicistronic DNA vaccines encoding OmpK gene and CC-motif ligand 4 gene(p-OmpK-CCL4),or Ompk gene and CC-motif ligand 19 gene(p-OmpK-CCL19)were successfully constructed.The results showed that two bicistronic DNA vaccines expressed Ompk protein of Vibrio anguillarum and CCL4/CCL19 proteins of fl ounder both in vitro and in vivo.After immunization,a large number of leucocytes in muscle were recruited at the injection site in treatment groups.The constructed vaccines induced signifi cant increases in CD4-1^(+) and CD4-2^(+) T lymphocytes,and sIgM^(+) B lymphocytes in peripheral blood,spleen,and head kidney.The percentage of T lymphocytes peaked on the 14^(th) post-vaccination day whereas that of B lymphocytes peaked in the 6^(th) post-vaccination week.Moreover,the expression profi les of 10 immune-related genes increased in muscles around the injection site,spleen,and head kidney.After the challenge,p-OmpK-CCL4 and p-OmpK-CCL19 conferred a relative percentage survival(RPS)of 74.1%and 63.3%,respectively,higher than p-OmpK alone(40.8%).In conclusion,both CCL4 and CCL19 can improve the protection of p-OmpK via evoking local immune response and then humoral and cellular immunity.CCL4 and CCL19 will be potential molecular adjuvants for use in DNA vaccines.

Covid-19 Mutations and the Effect of Different Vaccines on Immune Memory

We traced the coronavirus classification and evolution,analyzed the Covid-19 composition and its distinguishing characteristics when compared to SARS-CoV and MERS-CoV.Despite their close kinship,SARS-CoV and Covid-19 display significant structural differences,including 380 amino acid substitutions,and variable homology between certain open reading frames that are bound to diversify the pathogenesis and virulence of the two viral compounds.A single amino acid substitution such as replacing Aspartate(D)with Glycine(G)composes the D614G mutation that is around 20%more infectious than its predecessor 614D.The B117 variant,that exhibits a 70%transmissibility rate,harbours 23 mutants,each reflecting one amino acid exchange.We examined several globally spreading mutations,501.V2,B1351,P1,and others,with respect to the specific amino acid conversions involved.Unlike previous versions of coronavirus,where random mutations eventually precipitate extinction,the multiplicity of over 300,000 mutations appears to have rendered Covid-19 more contagious,facilitating its ability to evade detection,thus challenging the effectiveness of a large variety of emerging vaccines.Vaccination enhances immune memory and intelligence to combat or obstruct viral entry by generating antibodies that will prohibit the cellular binding and fusion with the Spike protein,restricting the virus from releasing its contents into the cell.Developing antibodies during the innate response,appears to be the most compelling solution in light of the hypothesis that Covid-19 inhibits the production of Interferon type I,compromising adaptive efficiency to recognize the virus,possibly provoking a cytokine storm that injures vital organs.With respect to that perspective,the potential safety and effectiveness of different vaccines are evaluated and compared,including the Spike protein mRNA version,the Adenovirus DNA,Spike protein subunits,the deactivated virus genres,or,finally,the live attenuated coronavirus that appears to demonstrate the greatest effectiveness,yet,encompass a relatively higher risk.

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

H5N1 鸟的流行性感冒病毒(A/chicken/Hubei/489/2004 ) 的 M 和 NP 基因被 RT-PCR 从病毒的 RNA 放大，并且分别地克隆向量进 pMD18-T。包含 M 基因(pHM6-m ) 或 NP 基因(pHM6-np ) 的表示 plasmid 然后被把 M 或 NP 基因插入到 pHM6 优核质表示向量构造；构造 plasmid 然后被定序。32 只 BALB/c 老鼠(6-week-old ) 在随机被划分成四个组。三组 BALB/c 老鼠被接种一次有 plasmid pHM6-m， plasmid pHM6-np 的 30 渭 g 或 plasmid pHM6-m (15 渭 g ) 和 pHM6-np (15 渭 g ) 的混合的任何一个 30 渭 g 的肌内的线路分别地。老鼠的一个另外的组作为控制与 100 渭 l PBS 被注射。二个星期以后，所有老鼠与相应 H5N1 鸟的流行性感冒病毒被质问，并且在下列 12 天内观察了。在 pHM6-m 组， pHM6-np 组和混合 plasmids 组的老鼠的幸存率分别地是 62.5% ， 25.0% 和 50.0% 。结果证明有效保护能被 pHM6-m 或 pHM6-np 提供，但是 pHM6-m 比 pHM6-np 提供了更好保护的效果。关键词 H5N1 流行性感冒病毒 - M 基因 - NP 基因 - 克隆 - DNA 疫苗的 CLC 数字 S852.65 基础条款：国家基本科学才能训练资助(NSFC J0630648 )

Immunization with Cytomegalovirus Envelope Glycoprotein M and Glycoprotein N DNA Vaccines can Provide Mice with Complete Protection against a Lethal Murine Cytomegalovirus Challenge

Human cytomegalovirus virions contain three major glycoprotein complexes (gC I, II, III), all of which are required for CMV infectivity. These complexes also represent major antigenic targets for anti-viral immune responses. The gC II complex consists of two glycoproteins, gM and gN. In the current study, DNA vaccines expressing the murine cytomegalovirus (MCMV) homologs of the gM and gN proteins were evaluated for protection against lethal MCMV infection in a mouse model. Humoral and cellular immune responses, spleen viral titers, and mice survival and body-weight changes were examined. The results showed that immunization with gM or gN DNA vaccine alone was not able to offer good protection, whereas co-immunization with both gM and gN induced an effective neutralizing antibody response and cellular immune response, and provided mice with complete protection against a lethal MCMV challenge. This study provides the first in vivo evidence that the gC II (gM-gN) complex may be able to serve as a protective subunit antigen for future HCMV vaccine development.

DNA vaccines targeting amyloid-βoligomer ameliorate cognitive deficits of aged APP/PS1/tau triple-transgenic mouse models of Alzheimer’s disease

The amyloid-β(Aβ)oligomer,rather than the Aβmonomer,is considered to be the primary initiator of Alzheimer’s disease.It was hypothesized that p(Aβ3-10)10-MT,the recombinant Aβ3-10 gene vaccine of the Aβoligomer has the potential to treat Alzheimer’s disease.In this study,we intramuscularly injected the p(Aβ3-10)10-MT vaccine into the left hindlimb of APP/PS1/tau triple-transgenic mice,which are a model for Alzheimer’s disease.Our results showed that the p(Aβ3-10)10-MT vaccine effectively reduced Aβoligomer levels and plaque deposition in the cerebral cortex and hippocampus,decreased the levels tau protein variants,reduced synaptic loss,protected synaptic function,reduced neuron loss,and ameliorated memory impairment without causing any cerebral hemorrhaging.Therefore,this novel DNA vaccine,which is safe and highly effective in mouse models of Alzheimer’s disease,holds a lot of promise for the treatment of Alzheimer’s disease in humans.

DNA Vaccines Approach:From Concepts to Applications

DNA vaccines are the third generation vaccines based on purified plasmid preparations containing transgenes that encode antigenic/therapeutic proteins or peptides capable of triggering an immune response against a wide range of diseases. This vaccine platform presents several attributes that confer distinct advantages over other vaccine technologies in terms of safety, ease of fabrication and stability. Many aspects, such as antigen expression and especially vector design, are under study because of their great influence on immunogenicity and efficacy of DNA vaccines. In this regard, with the attempt of improving the efficiency of DNA vaccines, co-expression of stimulatory sequences and diverse vector delivery systems are being optimized. With this in mind, this review aims to giving a conceptual approach of DNA vaccines, explaining their mechanisms of action and listing the already licensed veterinary DNA vaccines presented in the market.

THE HUMORAL AND CELLULAR IMMUNE RESPONSES INDUCED BY HPV18L1-E6/E7 DNA VACCINES IN MICE

Objective To construct eukaryotic expression vector of HPV18 L1-E6, E7 chimeric gene and examine the humoral and cellular immune responses induced by this DNA vaccines in mice. Methods The C-terminal of major capsid protein L1 gene and mutant zinc finger domains of early E6/7 oncogenes in HPV18 were integrated and inserted into eukaryotic expression vector pVAX1 to generate vaccines pVAX1-L1E6Mxx, E7Mxx. CHO cells were transiently transfected with the individual construct. Target protein expressions in the lysate of the transfected cells were measured by ELISA and immunocytochemistry. After BALB/c mice were vaccinated with various recombinant plasmids(pVAX1-L1-E6M3 or pVAX1-L1-E7M3) and immunie adjuvants (pLXHDmB7-2 or LTB) through different administration routes (intramuscular or intranasal) , the great cellular immune responses were produced as revealed by delayed-type hypersensitivity (DTH) and lymphocyte proliferation, and the expression of IL-4 and IFN-γ cells in CD4 + and CD8 + subpopulations. Results The highly efficient expression of pVAX1-L1E6Mxx, E7Mxx vector in host eukaryotic cells were demonstrated both by ELISA and immunocytochemistry. The level of specific serum IgG against HPV in experiment groups mice was much higher than that of control group, and intranuscular immunization group had the highest antibody level. Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8+ IFN-γ + cells number, but CD4 + IL4 + cell number was higher in intranasal immunization groups. The immunization groups using pLXHDmB7-2 as adjuvant were superior to other groups in immunoresponse. Conclusion These DNA vaccines produce remarkable cellular and humoral immune responses in the mouse and may provide as prophylatic and therapeutic candidates for HPV induced cancer treatment.

Vaccines’ Safety and Effectiveness in the Midst of Covid-19 Mutations

We examined the coronavirus classification and evolution through its multiple mutations that have increased its transmissibility rate up to 70% globally, threatening to undermine the promise of a number of emerging vaccines that primarily focus on the immune detection of the Spike trimer. The safety and effectiveness of different vaccination methods are evaluated and compared, including the mRNA version, the Adenovirus DNA, Spike protein subunits, the deactivated virus genres, and the live attenuated coronavirus. Mutations have been long considered as random events, or mistakes during the viral RNA replication. Usually, what can go wrong will go wrong;therefore, repeated transformations lead to the extinction of a virus. On the contrary, the aggregate result of over 300,000 Covid-19 variants has expanded its transmissibility and infectiousness. Covid-19 mutations do not degrade the virus;they empower and facilitate its disguise to evade detection. Unlike other coronaviruses, Covid-19 amino acid switches do not reflect the random unfolding of errors that eventually eradicate the virus. Covid-19 appears to use mutations adaptively in the service of its survival and expansion. We cite evidence that Covid-19 inhibits the interferon type I production, compromising adaptive immunity from recognizing the virus. The deleterious consequences of the cytokine storm where the CD8+ killer cells injure the vital organs of the host may well be a Covid-19 manoeuvring to escape exposure. It is probable that evolution has programmed Covid-19 with an adeptness designed to debilitate key systemic defences to secure its subsistence. To date the infectiousness of the Covid-19 pandemic is exponentially increasing, denoting the possibility of an even more dangerously elusive, inconspicuous, and sophisticated version of the disease.

Rabies DNA Vaccines: Current Status and Future

Rabies continues to be a significant cause of human and animal mortality, despite the availability of safe and effective prophylactics. Apart from limited access, the cost and complex schedules of rabies biologics often impact on the success of post-exposure prophylaxis in humans in the endemic countries. Mass vaccination of dogs, critical in rabies control, often fails to achieve its goal in rabies-endemic countries due to logistic, animal and vaccine-related issues. DNA vaccination has been proposed as a cheaper and efficient strategy for rabies prophylaxis, and its feasibility has been demonstrated in a number of animal models including companion animals, since 1994. Despite the proven efficacy, the technology suffers from a few drawbacks that limit its large-scale application, such as delayed and weaker immune responses in larger animals. Recent advances in the field of vector design and delivery hold promise for enhancement of rabies DNA vaccine efficacy. The present article provides an overview of developments in the field of DNA rabies vaccination and its future prospects.

In Vivo Transfection Methods of DNA Vaccines

Advantages and disadvantages of four transfection methods of DNA vaccines into the body were introduced in this study, including direct transfection, transfection of bacterial vectors, cationic lipofection and cationic polymer transfection. Cationic polymer had the characteristics of high transfection efficiency, simple operation, wide application, good repeatability and low cell virulence, which could be expected as a new kind of effi- cient transfection reagents. In practical application, various transfection methods have their own advantages and disadvantages, so the best choice should be made in the design process of vaccines based on targets, test cost, using objects and its convenience.

Nucleic acid vaccines: A taboo broken and prospect for a hepatitis B virus cure

Although a prophylactic vaccine is available,hepatitis B virus(HBV)remains a major cause of liver-related morbidity and mortality.Current treatment options are improving clinical outcomes in chronic hepatitis B;however,true functional cure is currently the exception rather than the rule.Nucleic acid vaccines are among the emerging immunotherapies that aim to restore weakened immune function in chronically infected hosts.DNA vaccines in particular have shown promising results in vivo by reducing viral replication,breaking immune tolerance in a sustained manner,or even decimating the intranuclear covalently closed circular DNA reservoir,the hallmark of HBV treatment.Although DNA vaccines encoding surface antigens administered by conventional injection elicit HBVspecific T cell responses in humans,initial clinical trials failed to demonstrate additional therapeutic benefit when administered with nucleos(t)ide analogs.In an attempt to improve vaccine immunogenicity,several techniques have been used,including codon/promoter optimization,coadministration of cytokine adjuvants,plasmids engineered to express multiple HBV epitopes,or combinations with other immunomodulators.DNA vaccine delivery by electroporation is among the most efficient strategies to enhance the production of plasmid-derived antigens to stimulate a potent cellular and humoral anti-HBV response.Preliminary results suggest that DNA vaccination via electroporation efficiently invigorates both arms of adaptive immunity and suppresses serum HBV DNA.In contrast,the study of mRNA-based vaccines is limited to a few in vitro experiments in this area.Further studies are needed to clarify the prospects of nucleic acid vaccines for HBV cure.

HER2-Specific Vaccines for HER2-Positive Breast Cancer Immunotherapy

Anti-human epidermal growth factor receptor-2 (HER2) immunization can be elicited by vaccination with DNA encoding the extra- or intra-cellular domain (ECD or ICD) of HER2, naked or encap-sulated in viral vectors. HER2-peptides derived from ECD or ICD of HER2, and HER2-pulsed dendritic cells (DCs) or engineered DCs expressing HER2, respectively. We performed a computer- based literature search which includes but is not limited to the following keywords: breast cancer, immunotherapy, HER2-peptide vaccine, HER2-DNA vaccine, HER-DC vaccine, HER2 vaccine, and HER2/neu, in PubMed, Medline, EMBO and Google Scholar;data from recently reported clinical trials were also included. Drawing upon this synthesis of literature, this work summarizes the de-velopment and current trend in experimental and clinical investigations in HER2-positive breast cancer using HER2-specific vaccine and immunotherapy, focusing especially on: (i) DNA-;(ii) peptide-;and (iii) DC-based vaccines. It addresses interventions that have been applied to overcome immunotolerance thereby to improve treatment outcomes. These include blocking the inhibitory cytotoxic T lymphocyte-associated protein-4 (CTLA-4), which is expressed at high levels by regulatory T (Treg) cells, or complete Treg depletion to improve T-cell activation. Moreover, modulatory interventions can provide further improvement in the efficacy of HER2-specific vaccine. The interventions include the use of immunogenic adjuvants such as cytokines interleukin-12 (IL-12), tumor necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor (GM-CSF), the use of Toll-like receptor (TLR) ligands and tetanus toxin’s universal epitopes such as the CD4+ help T (Th)-epitope P30, and the use of either chimeric or heterogenous xenogeneic HER2. Combining active HER2-vaccination with adoptive trastuzumab antibody immunotherapy is likely to increase the effectiveness of each approach alone. The development of effective HER2-vaccines for breast cancer remains a critical challenge. Though these novel interventions seem promising, further investigation is still needed since the results are preliminary. Furthermore, the review discusses the challenges and future perspectives in HER2-vaccine research and development.

Using Bacterial Vectors for Probable Vaccines: From Molecular Mechanism to Cancer Therapy

Anti-cancer therapies over the few decades, faced with many challenges. And bacterial vaccine vectors have shown a potential to be replaced as the cutting-edge technology for such aspects. Bacterial vaccine vectors with a suitable DNA can be a potential option for cancer treatment as a carrier for tumoricidal agents or bacterially directed Enzyme Prodrug treatment. Throughout this study, it is planned to have a review of the use of bacteria as vehicles by different ways for cancer treatment, detailing the systems of function and achievements at preclinical and clinical levels.

Oral PLGA-based DNA vaccines using interferons as adjuvants remarkably promote the immune protection of grass carp(Ctenopharyngodon idella)against GCRV infection

Grass carp hemorrhagic disease caused by grass carp reovirus(GCRV)results in significant economic losses to the global grass carp aquaculture industry.Oral vaccination is an ideal choice for disease precaution in aquaculture.However,oral vaccine can be degraded in the gut.Therefore,the selection of loading materials is essential.In this study,the S6 and S7 fragments(encoding the outer capsid protein VP4 and fibronectin VP56 of GCRV)and grass carp interferons(IFNs),including IFN1,IFN3,and IFNγ2 were used to create DNA vaccines and adjuvants based on pcDNA3.1,respectively.The oral DNA vaccine was encapsulated in poly(lactic-co-glycolic acid)(PLGA)and polyvinyl alcohol(PVA)with IFNs.The PLGA-PVA(PP)nano-microspheres were prepared by double emulsionsolvent evaporation technique.Using transmission electron microscopy and dynamic light scattering assays,it was determined the vaccines had a spherical structure with uniform particle size(643.5±35.3 nm).The nanomicrospheres possessed excellent encapsulation efficiency(81.6±2.6%)and loading rate(0.54±0.02%),and simultaneously exhibited negligible hemolytic activity and cell toxicity.The protection rate and tissue viral loads post-GCRV challenge in grass carp were assessed.The oral PP nano-microsphere with pVP4 t pIFN1(PP41)vaccine increased protection rate by 44%compared with the control group and was correlated with relatively low viral loads in the spleen,head kidney,and hindgut.Further,three crucial serum biochemical indexes,total superoxide dismutase(TSOD),complement C3(C3),and lysozyme(LZM),were also dramatically increased.Furthermore,mRNA expressions of representative immune-related genes(IgM,IFN1,IFNγ2,MHC-I,and CD8α)in the head kidney and spleen were significantly enhanced.In addition,mRNA expression of IgT was significantly boosted in the hindgut.The results indicate that DNA vaccine capsulated with PP is effective against GCRV infection.The present study provides insights into a prospective strategy for oral vaccine development in aquaculture.

Comparison of sperm parameters and DNA fragmentation index between infertile men with infection and vaccines of COVID-19

Several preventive measures,including vaccination,have been implemented owing to the severe global effect of coronavirus disease 2019(COVID-19),but there is still limited evidence in the effect of this disease and vaccination against it on male fertility.Therefore,this study is to compare sperm parameters of infertile patients with or without COVID-19 infection and the effect of COVID-19 vaccine types on them.Semen samples of infertile patients were collected consecutively at Universitas Indonesia-Cipto Mangunkusumo Hospital(Jakarta,Indonesia).COVID-19 was diagnosed by rapid antigen or polymerase chain reaction(PCR)tests.Vaccination was performed with three types of vaccine,namely inactivated viral vaccine,messenger RNA(mRNA)vaccine,and viral vector vaccine.Spermatozoa were then analyzed on the World Health Organization recommendations,and DNA fragmentation was assayed with the sperm chromatin dispersion kit.The results showed that the COVID-19 group experienced a significant decrease in sperm concentration and progressive motility(both P<0.05),but there was no significant change in morphology or sperm DNA fragmentation index(DFI;both P>0.05).The viral vector vaccine caused a decrease in morphology as well as an increase in DFI compared with the control(both P<0.05),meanwhile results for those who were vaccinated with the inactivated and mRNA types were not significant compared with the control(both P>0.05).We conclude that COVID-19 has negative effects on sperm parametes and sperm DNA fragmentation,and we found that the viral vector vaccines affect sperm parameter values and DNA fragmentation negatively.Further studies with a larger population and longer follow-up are needed to confirm the results.

Construction of multivalent DNA vaccines for Mycobacte-rium tuberculosis and its immunogenicity

The coding regions of Ag85B MPT-64, and ESAT-6 secreted proteins were cloned initially into the eu-karyotic expression vector pJW4303, then transformed to E. coli Top 10 strain for plasmid DNA extraction and further analysis. Plasmids containing the right insertion were se-quenced to confirm their identity. COS7 cells were trans-fected with a mixture containing serially diluted plasmid DNA encoding three secreted proteins and Lipofectin (Gibco). The supernatants and pellets prepared from various cell lines were run on SDS-PAGE gel and the expression of these proteins in COS7 cells were demonstrated by immunoblot using polyclonal or monoclonal antiserum of M.TBH37Rv. 21 days after first vaccination of C57BL-6 mice by all three recombinant eukaryotic expressing vectors, antibody titer for Ag85B reached 1:3200. 21 days after second vaccination, the antibody titer reached 1:102400. The highest antibody levels induced by multivalent vaccines after the second injection were equal to or even greater than the

Specific cellular immune responses in mice immunized with DNA,adeno-associated virus and adenoviral vaccines of Epstein-Barr virus-LMP2 alone or in combination

Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persistent Epstein-Barr virus (EBV) infection.NPC vaccine studies have focused on enhancing specific antiviral CTL responses.In this study,three vaccines capable of expressing the EBV-latent membrane protein 2 (LMP2) (a DNA vector,an adeno-associated virus (AAV) vector,and a replication-defective adenovirus serotype 5 (Ad5) vector) were respectively used to immunize female Balb/c mice (4-6 weeks old) at weeks 0,2 and 4,either alone or in combination.Our results suggest that combined immunization with DNA,AAV,and adenovirus vector vaccines induced specific cellular immunity more effectively than any of these vectors alone or a combination of two of the three,constituting a sound vaccine strategy for the prevention and treatment of NPC.

Hepatitis E virus chimeric DNA vaccine elicits immunologic response in mice

AIM： To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice. METHODS： The gene encoding the structural protein of HEV ORF2 fragment and full-length ORF3 was amplified by PCR. The PCR products were cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly thrice at a dose of 100 or 200 ug. Mice injected with empty pcDNA3 DNA or saline served as control and then specific immune responses in the mice were detected. RESULTS： After 2-3 times of inoculation, all mice injected with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte proliferation. The lymphocyte stimulation index in the group immunized with pcHEV23 （3.1＋0.49） was higher than that in the control group （0.787±0.12, P〈0.01）. None in the control group had a detectable level of anti-HEV IgG. CONCLUSION： DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully induce specific humoral and cellular immune response in mice.