Dendritic Cell-Directed Vaccination with a Lentivector Encoding PSCA for Prostate Cancer in Mice

Many studies have demonstrated that prostate stem cell antigen (PSCA) is an attractive target for immunotherapy based on its overexpression in prostate tumor tissue, especially in some metastatic tissues. In this study, we evaluated dendritic cell (DC)-directed lentiviral vector (DCLV) encoding murine PSCA (DCLV-PSCA) as a novel tumor vaccine for prostate cancer in mouse models. We showed that DCLV-PSCA could preferentially deliver the PSCA antigen gene to DC-SIGN-expressing 293T cells and bone marrow-derived DCs (BMDCs). Direct immunization with the DCLV-PSCA in male C57BL/6 mice elicited robust PSCA-responsive CD8⁺ and CD4⁺ T cells in vivo. In a transgenic adenocarcinoma mouse prostate cell line (TRAMP-C1) synergetic tumor model, we further demonstrated that DCLV-PSCA-vaccinated mice could be protected from lethal tumor challenge in a prophylactic model, whereas slower tumor growth was observed in a therapeutic model. This DCLV-PSCA vaccine also showed efficacy in inhibiting tumor metastases using a PSCA-expressing B16-F10 model. Taken together, these data suggest that DCLV is a potent vaccine carrier for PSCA in delivering anti-prostate cancer immunity.     

Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of Mycobacterium tuberculosis heat shock protein 70 gene to an antigen gene

Recently, self-replicating RNA vaccines (RNA replicons) have emerged as an effective strategy for nucleic acid vaccine development. Unlike naked DNA vaccines, RNA replicons eventually cause lysis of transfected cells and therefore do not raise the concern of integration into the host genome. We evaluated the effect of linking human papillomavirus type 16 E7 as a model Ag to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of Ag-specific immunity generated by a Sindbis virus self-replicating RNA vector, SINrep5. Our results indicated that this RNA replicon vaccine containing an E7/HSP70 fusion gene generated significantly higher E7-specific T cell-mediated immune responses in vaccinated mice than did vaccines containing the wild-type E7 gene. Furthermore, our in vitro studies demonstrated that E7 Ag from E7/HSP70 RNA replicon-transfected cells can be processed by bone marrow-derived dendritic cells and presented more efficiently through the MHC class I pathway than can wild-type E7 RNA replicon-transfected cells. More importantly, the fusion of HSP70 to E7 converted a less effective vaccine into one with significant potency against E7-expressing tumors. This antitumor effect was dependent on NK cells and CD8(+) T cells. These results indicated that fusion of HSP70 to an Ag gene may greatly enhance the potency of self-replicating RNA vaccines.     

Detection of progeny immune responses after intravenous administration of DNA vaccine to pregnant mice

A number of factors influence the development of tolerance, including the nature, concentration and mode of antigen presentation to the immune system, as well as the age of the host. The studies were conducted to determine whether immunizing pregnant mice with liposome-encapsulated DNA vaccines had an effect on the immune status of their offspring. Two different plasmids (encoding antigens from HIV-1 and influenza virus) were administered intravenously to pregnant mice. At 9.5 days post conception with cationic liposomes, injected plasmid was present in the tissues of the fetus, consistent with trans-placental transfer. When the offspring of vaccinated dams were immunized with DNA vaccine, they mounted stronger antigen-specific immune responses than controls and were protected against challenge by homologous influenza virus after vaccination. Moreover, such immune responses were strong in the offspring of mothers injected with DNA plasmid 9.5 days after coitus. These results suggest that DNA vaccinated mothers confer the antigen-specific immunity to their progeny. Here we describe the methods in detail as they relate to our previously published work. Published: April 23, 2002     

表达人前列腺干细胞抗原小鼠前列腺癌模型的建立

目的探讨并建立可供药物评价或生物学功能研究的表达人PSCA抗原的小鼠肿瘤模型。方法克隆人PSCA基因,构建pcDNA-PSCA质粒,稳定转染RM-1细胞,用RT-PCR和流式检测的方法筛选稳定表达人PSCA抗原的RM-PSCA细胞株;再将RM-PSCA细胞接种C57BL/6小鼠,观察其致瘤性,并寻找能够稳定致瘤的细胞数量;进而观测RM-PSCA所致肿瘤的生长情况及小鼠存活状况。结果筛选到了表达人PSCA抗原的RM-PSCA细胞,且1×105个肿瘤细胞能够保证10只实验小鼠全部成瘤;所致肿瘤生长迅速,接种后小鼠的平均存活时间为37 d。结论该研究成功的建立了稳定表达人PSCA抗原的小鼠肿瘤模型。     

N-terminally fusion of Her2/neu to HSP70 decreases efficiency of Her2/neu DNA vaccine

DNA vaccines consisted of tumor-associated antigen (TAA) are well suited for immunotherapy against tumor. The construct can contain TAA fused to an appropriate molecule (biologic adjuvant) to improve the efficacy of anti-tumor immune response. Heat shock protein 70 (HSP70) has been shown to be an excellent candidate, capable of cross-priming TAA by antigen presenting cells leading to a robust T-cell response. However, the relationship between strong T-cell responses and tumor rejection is not always mutually exclusive, for which TAA loss or activation of suppressive mechanisms may occur. HSP70 fused to downstream of Her2/neu as DNA vaccine has been shown to be efficient against Her2-expressing tumors. In this study, we examined if N-terminally fusion of Her2/neu to HSP70 could also improve efficiency of Her2/neu DNA vaccine. Therefore, mice with an established Her2/neu expressing tumor were immunized with DNA vaccine consisting of extracellular and trans-membrane domain (EC+TM) of rat Her2/neu alone or N-terminally fused to HSP70 and immune response was evaluated. Administration of rat Her2/neu led to partial control of tumor progression. Surprisingly, fusion of HSP70 to N-terminal of rat Her2/neu led to tumor progression. Our result proposes that fusion direction of biologic adjuvant is an important consideration when Her2/neu is used.     

Immunogenicity and Efficacy of Single Antigen Gp63, Polytope and PolytopeHSP70 DNA Vaccines against Visceral Leishmaniasis in Experimental Mouse Model

Polytope approach of genetic immunization is a promising strategy for the prevention of infectious disease as it is capable of generating effective cell mediated immunity by delivering the T cell epitopes assembled in series. Leishmaniasis is a significant world wide health problem for which no vaccine exists. In this study we have compared immunogenicity and efficacy of three types of DNA vaccines: single antigen Gp63 (Gp63/pcDNA), polytope (Poly/pcDNA) and Polytope fused with hsp70 (Poly/hsp/pcDNA) against visceral leishmaniasis in susceptible BALB/c mice. Mice vaccinated with these plasmids generated strong Th1 immune response as seen by dominating IFN-γ over IL-10 cytokine. Interestingly, cytotoxic responses generated by polytope DNA plasmid fused with hsp70 of Leishmania donovani were significantly higher when compared to polytope and single antigen Gp63 vaccine. Challenge studies revealed that the parasite load in liver and spleen was significantly lower with Poly/hsp/pcDNA vaccination compared to other vaccines. Therefore, our study indicates that polytope DNA vaccine is a feasible, practical and effective approach for visceral leishmaniasis.     

Heat shock protein 70 / MAGE-1 tumor vaccine can enhance the potency of MAGE-1–specific cellular immune responses in vivo

The cancer-testis antigen encoded by the MAGE-1 gene is an attractive antigen in tumor immunotherapy because it can be processed as a foreign antigen by the immune system and generate tumor-specific cellular immune response in vivo. However, increase of the potency of MAGE-1 DNA vaccines is still needed. The high degree of sequence homology and intrinsic immunogenicity of heat shock protein 70 (HSP70) have prompted the suggestion that HSP70 might have immunotherapeutic potential, as HSP70 purified from malignant and virally infected cells can transfer and deliver antigenic peptides to antigen-presenting cells to elicit peptide-specific immunity. In this research, we evaluated the enhancement of linkage of Mycobacterium tuberculosis HSP70 to MAGE-1 gene of the potency of antigen-specific immunity elicited by naked DNA vaccines. We found that vaccines containing MAGE-1-HSP70 fusion genes enhanced the frequency of MAGE-1–specific cytotoxic T cells in contract to vaccines containing the MAGE-1 gene alone. More importantly, the fusion converted a less effective DNA vaccine into one with significant potency against established MAGE-1–expressing tumors. These results indicate that linkage of HSP70 to MAGE-1 gene may greatly enhance the potency of DNA vaccines, and generate specific antitumor immunity against MAGE-1–expressing tumors.     

Toll-like receptor adaptor molecules enhance DNA-raised adaptive immune responses against influenza and tumors through activation of innate immunity

Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates the innate and adaptive immunity. TLR adaptor molecules play a central role in this cascade; thus, we hypothesized that overexpression of TLR adaptor molecules could mimic infection without any microbial components. Dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the Toll-interleukin-1 receptor domain-containing adaptor-inducing beta interferon (TRIF) or myeloid differentiation factor 88 (MyD88) were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection. These results indicate that TLR adaptor molecules can bridge innate and adaptive immunity and potentiate the effects of DNA vaccines against virus infection and tumors.     

Prevention of human PC-346C prostate cancer growth in mice by a xenogeneic tissue vaccine

Vaccination, as an approach to prostate cancer, has largely focused on immunotherapy utilizing specific molecules or allogeneic cells. Such methods are limited by the focused antigenic menu presented to the immune system and by immunotolerance to antigens recognized as “self”. To examine if a xenogeneic tissue vaccine could stimulate protective immunity in a human prostate cancer cell line, a vaccine was produced by glutaraldehyde fixation of harvested PAIII prostate cancer cells tumors (GFT cell vaccine) from Lobund-Wistar rats. Immunocompetent Ncr-Foxn1<nu> mice were vaccinated with the GFT cell vaccine four times, 7 days apart. The control animals were either not vaccinated or vaccinated with media or glutaraldehyde-fixed PC346C human prostate cancer cells and adjuvant. About 8 days after the final boost, serum and spleens were harvested. The splenocytes were co-incubated with PC346C cells and then transplanted orthotopically into sygneneic immunodeficient nude mice. About 10 weeks later, the prostates were weighed and sampled for histolologic examination. The spleens were harvested from additional mice, and the splenocytes were cultured, either with or without pulsing by GFT cells, and the supernatants harvested 72 h later for cytokine analysis. Results showed that vaccination with GFT cells resulted in increased serum antibody to a PAIII cell lysate; reduced weight of the prostate/seminal vesicle complex and reduced incidence of prostate cancer in nude mice; increased splenocyte supernatant levels of TNF-α, IL-2, IFN-γ and IL-12, cytokines associated with Th1 immunity; and increased splenocyte supernatant levels of IL-4 and IL-10, cytokines associated with Th2 immunity. In summary, the results suggest that use of a xenogeneic tissue vaccine can stimulate protective immunity against human prostate cancer cells.     

Characterization of HPV-16 E6 DNA vaccines employing intracellular targeting and intercellular spreading strategies

Summary Human papillomavirus (HPV) E6 and E7 are consistently expressed and are responsible for the malignant transformation of HPV-associated lesions. Thus, E6 and E7 represent ideal targets for therapeutic HPV vaccine development. We have previously used the gene gun approach to test several intracellular targeting and intercellular spreading strategies targeting HPV-16 E7. These strategies include the use of the sorting signal of lysosome-associated membrane protein (LAMP-1), Mycobacterium tuberculosis heat shock protein 70 (HSP70), calreticulin (CRT) and herpes simplex virus type 1 (HSV-1) VP22 proteins. All of these strategies have been shown to be capable of enhancing E7-DNA vaccine potency. In the current study, we have characterized DNA vaccines employing these intracellular targeting or intercellular spreading strategies targeting HPV-16 E6 for their ability to generate E6-specific CD8⁺ T cell immune responses and antitumor effects against an E6-expressing tumor cell line, TC-1, in C57BL/6 mice. We found that all the intracellular targeting strategies (CRT, LAMP-1, HSP70) as well as the intercellular spreading strategy (VP22) were able to enhance E6 DNA vaccine potency, although the orientation of HSP70 linked to E6 antigen in the E6 DNA vaccine appears to be important for the HSP70 strategy to work. The enhanced E6-specific CD8⁺ T cell immune response in vaccinated mice also translated into potent antitumor effects against TC-1 tumor cells. Our data indicate that all of the intracellular targeting and intercellular spreading strategies that have been shown to enhance E7 DNA vaccine potency were also able to enhance E6 DNA vaccine potency.     

Enhancement of DNA vaccine potency through linkage of antigen gene to ER chaperone molecules,ER-60, tapasin,and calnexin

DNA vaccines have emerged as an attractive approach for generating antigen-specific immunotherapy. Strategies that enhance antigen presentation may potentially be used to enhance DNA vaccine potency. Previous experiments showed that chimeric DNA vaccines utilizing endoplasmic reticulum (ER) chaperone molecules, such as Calreticulin (CRT), linked to an antigen were capable of generating antigen-specific CD8⁺ T cell immune responses in vaccinated mice. In this study, we tested DNA vaccines encoding the ER chaperone molecules ER-60, tapasin (Tap), or calnexin (Cal), linked to human papillomavirus type 16 (HPV-16) E7 for their abilities to generate E7-specific T cell-mediated immune responses and antitumor effects in vaccinated mice. Our results demonstrated that vaccination with DNA encoding any of these chaperone molecules linked to E7 led to a significant increase in the frequency of E7-specific CD8⁺ T cell precursors and generated stronger antitumor effects against an E7-expressing tumor in vaccinated mice compared to vaccination with wild-type E7 DNA. Our data suggest that DNA vaccines employing these ER chaperone molecules linked to antigen may enhance antigen-specific CD8⁺ T cell immune responses, resulting in a significantly more potent DNA vaccine.     

Plasmid DNA vaccine encoding prostatic acid phosphatase is effective in eliciting autologous antigen-specific CD8+ T cells

Prostatic acid phosphatase (PAP) is a prostate cancer tumor antigen and a prostate-specific protein shared by rats and humans. Previous studies indicated that Copenhagen rats immunized with a recombinant vaccinia virus expressing human PAP (hPAP) developed PAP-specific cytotoxic T cells (CTL) with cross reactivity to rat PAP (rPAP) and evidence of prostate inflammation. Viral delivery of vaccine antigens is an active area of clinical investigation. However, a potential difficulty with viral-based immunizations is that immune responses elicited to the viral vector might limit the possibility of multiple immunizations. In this paper, we investigate the ability of another genetic immunization method, a DNA vaccine encoding PAP, to elicit antigen-specific CD8+ T cell immune responses. Specifically, Lewis rats were immunized with either a plasmid DNA-based (pTVG-HP) or vaccinia-based (VV-HP) vaccine each encoding hPAP. We determined that rats immunized with a DNA vaccine encoding hPAP developed a Th1-biased immune response as indicated by proliferating PAP-specific CD4+ and CD8+ cells and IFNγ production. Rats immunized with vaccinia virus encoding PAP did not develop a PAP-specific response unless boosted with a heterologous vaccination scheme. Most importantly, multiple immunizations with a DNA vaccine encoding the rat PAP homologue (pTVG-RP) could overcome peripheral self-tolerance against rPAP and generate a Th1-biased antigen-specific CD4+ and CD8+ T cell response. Overall, DNA vaccines provide a safe and effective method of generating prostate antigen-specific T cell responses. These findings support the investigation of PAP-specific DNA vaccines in human clinical trials.     

Her-2 DNA versus cell vaccine: immunogenicity and anti-tumor activity

Direct comparison and ranking of vaccine formulations in pre-clinical studies will expedite the identification of cancer vaccines for clinical trials. Two human ErbB-2 (Her-2) vaccines, naked DNA and whole cell vaccine, were tested side-by-side in wild type and Her-2 transgenic mice. Both vaccines can induce humoral and cellular immunity to the entire repertoire of Her-2 epitopes. Mice were electro-vaccinated i.m. with a mixture of pGM-CSF and pE2TM, the latter encodes Her-2 extracellular and transmembrane domains. Alternatively, mice were injected i.p. with human ovarian cancer SKOV3 cells that have amplified Her-2. In wild type mice, comparable levels of Her-2 antibodies (Ab) were induced by these two vaccines. However, T cell immunity and protection against Her-2⁺ tumors were superior in DNA vaccinated mice. In BALB Her-2 transgenic (Tg) mice, which were tolerant to Her-2, DNA and cell vaccines were administered after regulatory T cells (Treg) were removed by anti-CD25 mAb. Again, comparable levels of Her-2 Ab were induced, but DNA vaccines rendered greater anti-tumor activity. In B6xDR3 Her-2 Tg mice that expressed the autoimmune prone HLA-DR3 allele, higher levels of Her-2 Ab were induced by SKOV3 cell than by Her-2 DNA. But anti-tumor activity was still more profound in DNA vaccinated mice. Therefore, Her-2 DNA vaccine induced greater anti-tumor immunity than cell vaccine, whether mice were tolerant to Her-2 or susceptible to autoimmunity. Through such side-by-side comparisons in appropriate pre-clinical test systems, the more effective vaccine formulations will emerge as candidates for clinical trials. P. J. Whittington, O. Radkevich-Brown and J. B. Jacob have contributed equally to this work.     

Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines

The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8(+) T-cell and antibody responses by approximately 100- and approximately 1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIV env and HIV gag DNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of the env DNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.     

Improving Multi-Epitope Long Peptide Vaccine Potency by Using a Strategy that Enhances CD4+ T Help in BALB/c Mice

Peptide-based vaccines are attractive approaches for cancer immunotherapy; but the success of these vaccines in clinical trials have been limited. Our goal is to improve immune responses and anti-tumor effects against a synthetic, multi-epitope, long peptide from rat Her2/neu (rHer2/neu) using the help of CD4+ T cells and appropriate adjuvant in a mouse tumor model. Female BALB/c mice were vaccinated with P₅₊₄₃₅ multi-epitope long peptide that presents epitopes for cytotoxic T lymphocytes (CTL) in combination with a universal Pan DR epitope (PADRE) or CpG-oligodeoxynucleotides (CpG-ODNs) as a Toll-like receptor agonist adjuvant. The results show that vaccination with the multi-epitope long peptide in combination with the PADRE peptide and CpG-ODN induced expansion of subpopulations of CD4+ and CD8+ cells producing IFN-γ, the average tumor size in the vaccinated mice was less than that of the other groups, and tumor growth was inhibited in 40% of the mice in the vaccinated group. The mean survival time was 82.6 ± 1.25 days in mice vaccinated with P₅₊₄₃₅ + CpG+ PADRE. Our results demonstrate that inclusion of PADRE and CpG with the peptide vaccine enhanced significant tumor specific-immune responses in vaccinated mice.     

Vaccination with DNA Plasmids Expressing Gn Coupled to C3d or Alphavirus Replicons Expressing Gn Protects Mice against Rift Valley Fever Virus

Background

Rift Valley fever (RVF) is an arthropod-borne viral zoonosis. Rift Valley fever virus (RVFV) is an important biological threat with the potential to spread to new susceptible areas. In addition, it is a potential biowarfare agent.

Methodology/Principal Findings

We developed two potential vaccines, DNA plasmids and alphavirus replicons, expressing the Gn glycoprotein of RVFV alone or fused to three copies of complement protein, C3d. Each vaccine was administered to mice in an all DNA, all replicon, or a DNA prime/replicon boost strategy and both the humoral and cellular responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. Mice vaccinated with an inactivated form of MP12 did elicit high titer antibodies, but these antibodies were unable to neutralize RVFV infection. However, only vaccine strategies incorporating alphavirus replicons elicited cellular responses to Gn. Both vaccines strategies completely prevented weight loss and morbidity and protected against lethal RVFV challenge. Passive transfer of antisera from vaccinated mice into naïve mice showed that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited antibodies that protected mice as well as sera from mice immunized with MP12.

Conclusion/Significance

These results show that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn administered alone or in a DNA prime/replicon boost strategy are effective RVFV vaccines. These vaccine strategies provide safer alternatives to using live-attenuated RVFV vaccines for human use.     

Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines

Heterologous "prime-boost" regimens that involve priming with plasmid DNA vaccines and boosting with recombinant viral vectors have been shown to elicit potent virus-specific cytotoxic T-lymphocyte responses. Increasing evidence, however, suggests that the utility of recombinant viral vectors in human populations will be significantly limited by preexisting antivector immunity. Here we demonstrate that the coadministration of plasmid chemokines and colony-stimulating factors with plasmid DNA vaccines markedly increases the immunogenicity of DNA prime-recombinant adenovirus serotype 5 (rAd5) boost and DNA prime-recombinant vaccinia virus (rVac) boost vaccine regimens in BALB/c mice. In mice with preexisting anti-Ad5 immunity, priming with the DNA vaccine alone followed by rAd5 boosting elicited only marginal immune responses. In contrast, cytokine-augmented DNA vaccine priming followed by rAd5 vector boosting was able to generate potent immune responses in mice with preexisting anti-Ad5 immunity. These data demonstrate that plasmid cytokines can markedly improve the immunogenicity of DNA prime-viral vector boost vaccine strategies and can partially compensate for antivector immunity.     

前列腺干细胞抗原(PSCA)的表达及其特异结合肽的筛选

通过反转录 PCR从人前列腺癌细胞中克隆了前列腺干细胞抗原 (PSCA)基因 ,在大肠杆菌中利用pQE30载体对截断型PSCA基因进行了可溶性表达。蛋白纯化后 ,利用噬菌体随机展示 12肽库筛选了PSCA蛋白的特异结合肽 ,通过与EGFP蛋白的耦联表达验证了结合肽的特异性。此特异结合肽的获得 ,为进一步研究针对PSCA的前列腺癌靶向免疫治疗奠定了基础     

增强DNA疫苗免疫应答的新进展

DNA疫苗为编码抗原蛋白的真核表达载体,注入体内后在原位表达所编码的抗原并诱导免疫应答,在预防感染、治疗自身免疫性疾病、过敏性疾病和肿瘤等疫病中有着很好的应用前景。但与灭活疫苗相比,其免疫效价还比较低。有多种策略能够增强或调节DNA疫苗诱导的免疫应答,其中,作为外源基因载体的质粒的组成及插入的有关基因均可直接或间接地影响免疫反应的效果,在构建DNA疫苗质粒时,加入细胞因子、融合信号、泛素等基因以及ISS序列,另外还可以通过设计一些对抗原提成细胞有影响的分子共注射,以及加入转移分子,都可以明显增强DNA疫苗的免疫效果,从而有利于研制更有效的DNA疫苗。