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ABSTRACT: Abstract The attenuated or non-pathogenic live vectors have been evolved specifically to deliver DNA into cells as efficient delivery tools in gene therapy. Recently, a non-pathogenic protozoan, Leishmania tarentolae (L.tar) has attracted a great attention. In current study, we used Leishmania expression system (LEXSY) for stable expression of HPV16 E7 linked to different mini-chaperones [N-/C-terminal of gp96] and compared their immunogenicity and protective effects in C57BL/6 mice against TC-1 challenge. TC-1 murine model is primary C57BL/6 mice lung epithelial cells co-transformed with HPV16 E6, HPV16 E7 and ras oncogenes. Our results showed that subcutaneous administration of mice with both the recombinant L.tar-E7-NT (gp96) and L.tar-E7-CT (gp96) led to enhance the levels of IFN-γ and also IgG2a before and after challenge with TC-1. Furthermore, L.tar-E7-CT (gp96) live vaccine indicated significant protective effects as compared to control groups as well as group vaccinated with L.tar-E7. Indeed, the recombinant live vector is capable of eliciting effective humoral and cellular immune responses in mice, but however, further studies are required to increase their efficacy.
Drug Delivery 06/2013; · 1.46 Impact Factor
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Noushin Saljoughian,
Tahereh Taheri,
Farnaz Zahedifard,
Yasaman Taslimi,
Fatemeh Doustdari, Azam Bolhassani,
Delaram Doroud,
Hiva Azizi,
Kazem Heidari,
Mohammad Vasei,
Nabiollah Namvar Asl,
Barbara Papadopoulou,
Sima Rafati
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ABSTRACT: Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB(-CTE))) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB(-CTE)-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB(-CTE)-recombinant L. tarentolae as a safe live vaccine candidate against VL.
PLoS Neglected Tropical Diseases 04/2013; 7(4):e2174. · 4.69 Impact Factor
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ABSTRACT: Background: Cervical cancer, the third most prevalent cause of cancer in women worldwide, is associated with HPVs. The critical role of E7 protein in HPV-related malignancies has designated it as a strong contender for generating vaccines against HPV. Materials & methods: In this study, we developed a novel live vaccine using recombinant Leishmania tarentolae expressing E7-green fluorescent protein (GFP) fusion protein for the protection of mice against HPV-associated tumors. In order to transfect L. tarentolae with E7-GFP fusion construct, pLEXSY-neo2 system was applied. Followed by PCR, fluorescence imaging and fluorescence-activated cell sorting analysis, integration of E7-GFP gene into parasites genome was confirmed. A comparative study of six groups of C57BL/6 mice was performed to analyze antigen-specific humoral and cellular immune responses against E7 encoding live and DNA vaccines. Furthermore, the anti-tumor protective effect of L. tarentolae-E7-GFP was compared to other vaccination strategies, namely pcDNA-E7 as the DNA vaccine and pcDNA-E7/L. tarentolae-E7-GFP as the prime-boost regimen. Results: We found that E7-GFP expressing recombinant L. tarentolae induces significant levels of IgG2a and IFN-γ, while there is no significant IL-5 production compared with that of other strategies and control groups before and after challenge with TC-1 tumor cells. It is noteworthy that the designed live vaccine showed the best protection and minimum tumor size among all groups against TC-1-induced tumors. Conclusion: Overall, the results obtained revealed that the E7-GFP recombinant L. tarentolae could be a potential live vaccine for induction of immune responses in vivo.
Immunotherapy 11/2012; 4(11):1107-20. · 1.85 Impact Factor
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ABSTRACT: The immunogenic properties of heat shock proteins (HSPs) have prompted investigations into their application as immuno-modulatory agents. HSPs have been used as potent adjuvants in immunotherapy of cancer and infectious diseases. Some studies showed that immune activities reside within N- or C-terminal fragments of HSPs. These small fragments are sufficient to link peptides, to bind and be taken up by the receptors CD91 and scavenger receptor type A on antigen presenting cells (APCs). Thus, these mini-chaperones can be used in immunotherapy of tumors and vaccine development. The data clearly demonstrated the potential of using HSP fragments as a possible adjuvant to augment CTL response against infectious diseases. Some HSP domains have been shown to inhibit endothelial cell growth, angiogenesis or tumor growth. In this review, we describe the immuno-stimulatory activities of various mini-chaperones in development of different vaccine strategies (DNA-based vaccine and protein/peptide-based vaccines).
Human vaccines & immunotherapeutics. 10/2012; 9(1).
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ABSTRACT: DNA vaccines have emerged as a promising approach for generating antigen-specific immunotherapy. However, due to their low immunogenicity, there is a need to enhance DNA-based vaccine potency. Two main strategies to increase DNA-based vaccine potency are the employment of immuno-adjuvants such as heat shock proteins (HSPs) and a method of improving the delivery of naked plasmid DNA by electroporation. In the current study, we evaluated the effects of linkage of human papillomavirus (HPV) type 16 E7 as a model antigen to N-terminal and C-terminal of glycoprotein 96 (NT-/CT-gp96) on the potency of E7-specific immunity generated by DNA vaccines. We found that subcutaneous DNA injection with E7-CT (gp96) followed by electroporation generates the significant E7-specific IFN-γ immune responses as well as the best protective effects in vaccinated mice as compared to E7 or E7-NT (gp96) DNA vaccines. Therefore, our data indicate that subcutaneous administration of E7 DNA linked to CT (gp96) fragment followed by electroporation can significantly enhance the potency of DNA vaccines. Indeed, the structural domains of immuno-chaperones show the potential of generating effective immune responses against different clinical disorders such as cancer. Altogether, our results show that comparable regions of gp96 (N-/C-terminal fragments of gp96) may have qualitatively different immunological effects in vaccine design.
Immunology letters 10/2012; · 2.91 Impact Factor
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ABSTRACT: Although DNA vaccines represent an attractive approach for generating antigen-specific immunity, improvement of their potency is highly demanded. In the present study, three strategies including linkage to immunostimulatory molecules (N-terminal of gp96), co-administration of chemokines (IP-10 or RANTES) and PEI600-Tat as non-viral gene delivery system have been applied to enhance DNA vaccine efficacy against HPV infections. We found that C57BL/6 immunization with E7-NT-gp96 fusion gene led to increased level of IFN-γ compared to E7 alone. The fused genes showed considerable protective potency in tumor mice model. In addition, E7-NT-gp96 delivered with PEI600-Tat was more protective against E7-expressing tumors comparing with E7-NT-gp96 alone. Our results showed that co-administration of IP-10 with E7-NT-gp96 delivered by PEI600-Tat elicits significant IFN-γ production and consequently a strong preventive response against TC-1 tumor cells in contrast to increased tumor growth by RANTES co-delivery. Also in therapeutic experiment, our data showed that co-immunization of IP-10 at the same inoculation site of TC-1 along with E7-NT-gp96 delivery by PEI600-Tat is able to significantly suppress TC-1 tumor growth. The successful treatment by this immunization protocol was associated with the elevated levels of IFN-γ and IL-2 production in the lymph nodes. These data indicated that fusion of NT-gp96 to E7 in combination with IP-10 co-administration and PEI600-Tat delivery system can synergistically enhance the potency of HPV DNA vaccines. Therefore, this approach suggests a combinational therapeutic strategy against cervical and other HPV-related cancers.
Molecular Immunology 08/2012; 53(1-2):149-60. · 2.90 Impact Factor
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ABSTRACT: The design of efficient cancer treatments is one of the major challenges of medical science. Therapeutic vaccines of cancer have been emerged as an attractive approach for their capacity of breaking the immune tolerance and invoking long-term immune response targeting cancer cells without autoimmunity. An efficient antigen delivery system is the key issue of developing an effective cancer vaccine. In this regard, live vaccination strategies including various live bacterial and viral vectors have attracted a great attention. Several bacterial strains such as Salmonella, Listeria monocytogenes and Lactococcus lactis effectively colonize solid tumors and act as antitumor therapeutics. On the other hand, the use of viruses as vaccine vectors such as Vaccinia, Adenovirus, Herpes simplex virus, Paramyxovirus and Retroviruses utilizes mechanisms that evolved in these microbes for entering cells and capturing the cellular machinery to express viral proteins. Viral/bacterial-vectored vaccines induce systemic T-cell responses including polyfunctional cytokine-secreting CD4+ and CD8+ T-cells. However, there is an urgent need for the development of new safe live vaccine vectors that are capable of enhancing antigen presentation and eliciting potent immune responses without the risk of development of disease in humans. Recently, nonpathogenic parasites including Leishmania tarentolae, Toxoplasma gondii and Trypanosoma cruzi have emerged to be a novel candidate for gene delivery and heterologous genes expression. In this review, recent researches on cancer therapy using genetically modified bacteria and virus are summarized. In addition, live parasite-based vectors will be discussed as a novel anticancer therapeutic approach.
International Journal of Cancer 05/2012; 131(8):1733-43. · 5.44 Impact Factor
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11/2011; , ISBN: 978-953-307-538-9
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ABSTRACT: To control cervical cancer, efficient vaccination against human papillomavirus (HPV) is highly required. Despite the advantages and safety of the protein vaccines, additional strategies to enhance their immunogenicity are needed. E7 is a transforming protein which represents a perfect target antigen for vaccines or immunotherapies. Heat shock proteins (HSPs) facilitate cellular immune responses to antigenic peptides or proteins bound to them. Regarding to previous studies, vaccination with purified HSP/antigen complexes efficiently elicit antigen-specific immune responses in mice model. The N-terminal of glycoprotein 96 (NT-gp96) has adjuvant effect and can induce effective cumulative immune response against clinical disorders, especially cancers. In this study, the recombinant HPV16 E7 and E7 linked to NT-gp96 (E7-NT-gp96) proteins were generated in prokaryotic expression system. Mice were vaccinated twice with this recombinant proteins and the immunogenicity of the fusion protein was determined. The preventive efficacy of E7-NT-gp96 fusion protein was also evaluated and compared to E7 protein after challenging with cancerous TC-1 cell line. In vitro re-stimulated splenocytes of mice vaccinated with rE7-NT-gp96 protein induced higher IFN-γ response in comparison with E7 protein immunization. Moreover, immunization with E7-NT-gp96 protein displayed low but stable humoral responses at post-challenge time. The data showed that vaccination with fused E7-NT-gp96 protein delayed the tumour occurrence and growth as compared to protein E7 alone. These results suggest that fused adjuvant-free E7-NT-gp96 protein vaccination could direct the immune responses towards Th1 immunity. Furthermore, the linkage of NT-gp96 to E7 could enhance protective anti-tumour immunity.
Scandinavian Journal of Immunology 09/2011; 75(1):27-37. · 2.23 Impact Factor
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ABSTRACT: Stimulation of neutrophils may potentiate immunity to Leishmania major. CpG-containing oligodeoxynucleotide (ODN) has immune stimulatory effects and has been suggested as adjuvants and therapeutics to potentiate efficacy of vaccines and treatments against leishmaniasis. Here, we examined the stimulatory effect of synthetic ODN containing CpG motifs class A and B on cytokine production by neutrophils. Neutrophils from healthy donors responded to CpG-ODN type A, but not to class B, with secretion of IL-8 and following GM-CSF pretreatment with TNF-α production. To test whether neutrophil responses were altered in cutaneous leishmaniasis (CL) and to better understand the role of neutrophils in susceptibility and resistance to disease, we evaluated cytokine responses in GM-CSF preconditioned neutrophils from asymptomatic (Leishmanin skin test positive, LST+) and nonhealing CL individuals to CpG-ODN class A and assessed the expression levels of toll-like receptors (TLR2), 4 and 9. LST+ and healthy donor, but not nonhealing CL neutrophils, responded with TNF-α secretion. Neutrophils from nonhealing CL displayed increased mRNA expression levels of TLR2, 4 and 9 compared to neutrophils from LST+ or healthy donors. Therefore, failure to cure CL is associated with reduced ability of neutrophils to secrete TNF-α and correlates with high TLR 2, 4 and 9 expressions.
Parasite Immunology 07/2011; 33(11):609-20. · 2.60 Impact Factor
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ABSTRACT: Reporter genes have proved to be an excellent tool for studying disease progression. Recently, the green fluorescent protein (GFP) ability to quantitatively monitor gene expression has been demonstrated in different organisms. This report describes the use of Leishmania tarentolae (L. tarentolae) expression system (LEXSY) for high and stable levels of GFP production in different Leishmania species including L. tarentolae, L. major and L. infantum. The DNA expression cassette (pLEXSY-EGFP) was integrated into the chromosomal ssu locus of Leishmania strains through homologous recombination. Fluorescent microscopic image showed that GFP transgenes can be abundantly and stably expressed in promastigote and amastigote stages of parasites. Furthermore, flow cytometry analysis indicated a clear quantitative distinction between wild type and transgenic Leishmania strains at both promastigote and amastigote forms. Our data showed that the footpad lesions with GFP-transfected L. major are progressive over time by using fluorescence small-animal imaging system. Consequently, the utilization of stable GFP-transfected Leishmania species will be appropriate for in vitro and in vivo screening of anti-leishmanial drugs and vaccine development as well as understanding the biology of the host-parasite interactions at the cellular level.
Experimental Parasitology 03/2011; 127(3):637-45. · 2.12 Impact Factor
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ABSTRACT: Cancer vaccines are the promising tools in the hands of the clinical oncologist. Many tumor-associated antigens are excellent targets for immune therapy and vaccine design. Optimally designed cancer vaccines should combine the best tumor antigens with the most effective immunotherapy agents and/or delivery strategies to achieve positive clinical results. Various vaccine delivery systems such as different routes of immunization and physical/chemical delivery methods have been used in cancer therapy with the goal to induce immunity against tumor-associated antigens. Two basic delivery approaches including physical delivery to achieve higher levels of antigen production and formulation with microparticles to target antigen-presenting cells (APCs) have demonstrated to be effective in animal models. New developments in vaccine delivery systems will improve the efficiency of clinical trials in the near future. Among them, nanoparticles (NPs) such as dendrimers, polymeric NPs, metallic NPs, magnetic NPs and quantum dots have emerged as effective vaccine adjuvants for infectious diseases and cancer therapy. Furthermore, cell-penetrating peptides (CPP) have been known as attractive carrier having applications in drug delivery, gene transfer and DNA vaccination. This review will focus on the utilization of different vaccine delivery systems for prevention or treatment of cancer. We will discuss their clinical applications and the future prospects for cancer vaccine development.
Molecular Cancer 01/2011; 10:3. · 3.99 Impact Factor
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ABSTRACT: An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.
Experimental Parasitology 01/2011; 128(1):9-17. · 2.12 Impact Factor
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ABSTRACT: Cervical cancer is the second most frequent cancer among females worldwide, especially human papilloma viruses (HPV) types 16 and 18. In viral systems the identification of serological markers would facilitate the diagnosis of HPV infections and virus-related disease. The aim of the present investigation was to determine and search for serologic markers in cervical cancer patients associated with HPV.
A total of 58 Iranian women with invasive cervical carcinoma including adenocarcinoma and squamous cell carcinoma (SCC) were included. Serum antibody response to HPV infections in patients was detected by Western blot and ELISA techniques based on recombinant HPV16E7 and the N-terminal and C-terminal fragments of gp96 (NT-gp96 and CT-gp96) proteins. These recombinant proteins were expressed in Escherichia coli as a His-tag protein and purified using affinity chromatography.
The ELISA results indicated that patients with high antibody response to HPV16E7 had significant seroreactivity to CT-gp96 fragment. In Western blot analysis, a strong association between anti-E7, anti-NT-gp96 and anti-CT-gp96 reactivity and cervical cancer was obtained using purified recombinant proteins. In adenocarcinoma cases, no significant difference was observed in seroreactivities between normal and patients.
The evaluation of cervical cancer patients' seroreactivities against three recombinant proteins (rE7, rNT-gp96 and rCT-gp96) showed significantly higher levels of these markers in SCC only, but not in adenocarcinoma and control groups. Also, the usage of both techniques (ELISA and Western blotting) can provide more reliable tools for diagnosis of cervical cancer.
The Indian journal of medical research 11/2009; 130(5):533-41. · 1.84 Impact Factor
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ABSTRACT: Human papillomaviruses (HPVs) are simple, non-enveloped, double-stranded DNA viruses and responsible for an enormous global burden of genital disease. HPV is annually associated with 500,000 new cases of cervical cancer and 250,000 cervical cancer deaths worldwide. The association between HPV infection and cervical cancer indicates that HPV serves as an ideal target for development of preventive and therapeutic vaccines. A novel approach for primary prevention of cervical cancer has become available by the discovery of efficient prophylactic HPV vaccines based on virus-like particles. Therapeutic vaccination has been limited by inadequate antigen-specific immune responses. Different therapeutic strategies have been developed including peptide immunization-based therapies, DNA vector-based therapies, viral/bacterial vector-based therapies, immune response modifiers, photodynamic therapy (PDT) and T cell receptor based therapy. At present, the design of therapeutic vaccines to control the growth of HPV-induced tumors has focused on utilization of E6 and E7 proteins or peptides as vaccine antigens. Human trials are the most important test for the efficacy of HPV16/18 E6 and E7 proteins as immunotherapy for cervical cancer. This review attempts to describe different therapeutic vaccinations against HPV infections.
Human vaccines 11/2009; 5(10):671-89. · 3.58 Impact Factor
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ABSTRACT: DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. The mode of plasmid DNA delivery is critical to make progress in DNA vaccination. Using human papillomavirus type 16 E7 as a model antigen, this study evaluated the effect of peptide-polymer hybrid including PEI600-Tat conjugate as a novel gene delivery system on the potency of antigen-specific immunity in mice model. At ratio of 10:50 PEI-Tat/E7DNA (w/w), both humoral and cellular immune responses were significantly enhanced as compared with E7DNA construct and induced Th1 response. Therefore, this new delivery system could have promising applications in gene therapy.
Drug Delivery 06/2009; 16(4):196-204. · 1.46 Impact Factor
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ABSTRACT: Heat-shock proteins (HSPs) have been known as multifunctional proteins. They facilitate the folding and unfolding of proteins, participate in vesicular transport processes, prevent protein aggregation in the densely packed cytosol and are involved in signaling processes. HSPs have been involved in different fields, including autoimmunity, immunity to infections and tumor immunology. Although there are many different kinds of HSPs, only some HSPs, including HSP70 and Gp96, have immunological properties. HSP molecules have been applied into DNA- or protein (peptide)-based vaccines as antigens, chaperones or adjuvants. HSP-based vaccines have been shown to immunize against cancer and infectious diseases in both prophylactic and therapeutic protocols. The immunogenicity of HSPs results from two different properties: a peptide-dependent capacity to chaperone and elicit adaptive cytotoxic T-lymphocyte responses against antigenic peptides and a peptide-independent immunomodulatory capacity. Furthermore, HSPs could be immunoregulatory agents with potent and widely applicable therapeutic uses. Accordingly, certain HSPs, such as HSP70 and Gp96, are highly effective carrier molecules for cross-presentation. Their ability in eliciting immune responses against different pathogens (parasite and virus) and their role in cancer immunity will be discussed in this review.
Expert Review of Vaccines 11/2008; 7(8):1185-99. · 4.25 Impact Factor
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ABSTRACT: Human papillomavirus, particularly type 16 (HPV16) is present in more than 99% of cervical cancers. E7 is the major oncogenic protein produced in cervical cancer-associated HPV16. An efficient vaccine against viral infection requires induction of strong humoral and cellular responses against viral proteins. Heat shock proteins (HSPs) like Gp96 have been described as potent tumor vaccines in animal models and are currently studied in human clinical trials. In this study, we investigated the utility of HPV16 E7 along with Gp96 as an adjuvant in C57BL/6 mice model. We compared the level of humoral and cellular immune responses by E7+Gp96 co-injection as DNA/DNA and prime-boost (DNA/protein) immunization strategies. In prime-boost immunization strategies, we first immunized C57BL/6 mice with the complete open-reading frame of E7 and Gp96 (pcDNA-E7 and pcDNA-Gp96) and then boosted with rE7, rNT-gp96 (N-terminal extension of Gp96) and rCT-gp96 (C-terminal extension of Gp96) mixed with Montanide 720 in different formulations. The humoral immune responses against rE7 and the different truncated forms of rGp96 suggested a mixed Th1/Th2 response with high intensity toward Th2. Assessment of lymphoproliferative and cytokine responses against rE7 and the different fragments of Gp96, showed that DNA vaccination including E7 and Gp96 induced Th1 response. We concluded that co-delivery of naked DNA E7+Gp96 plasmid was immunologically more effective than E7 alone. Our study demonstrated that co-delivery of E7+Gp96 as DNA/DNA and E7+CT-gp96 as DNA/protein could be an effective approach to induce E7-specific immune responses as a potential vaccine candidate for cervical cancer.
Vaccine 07/2008; 26(26):3362-70. · 3.77 Impact Factor
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