Based On The Luciferase Activity Detected In Mice Challenged Model And The New Cationic Carriers For Dna Vaccine Mucosal Primary Effect

UNAIDS/WHO estimated that 2.7 million new HIV infections and 2 million HIV-related deaths occurred in 2007,currently～33 million people are living with HIV/AIDS.Although it is well known that an HIV/AIDS vaccine is the most effective approach to contain HIV epidemic,developing an effective vaccine against human HIV-1 remains elusive and faces great challenges after more than two decades of intensive research.One of those challenges is the difficulty to access suitable animal model for screening and prioritizing vaccine candidates.Since pigtail monkey and chimpanzee are the only animals infectable for HIV-1,the rarity and ethical concems exclude the usage of these animals to test vaccine efficacy.Currently the majority of HIV-1 preclinical vaccine efficacy studies are carried out in SHIV/Monkey model,however,highly cost and the regulatory requirement to use SHIV/Monkey model in P3 laboratory facility for large animals lead to unavailability of this model for the vast majority of researchers to test their vaccines or vaccine strategies.As known,HIV-1 is unable to infect small animals,the severe combined immunodeficiency(SCID) mice reconstituted with human derived PBMCs was used as small animal models for evaluating protective capacity of neutralizing antibodies and drugs efficacy.However, effective T-cell immunity cannot be raised in these models.In studyⅠ,we aim to establish a mice model for the evaluation of T-cell vaccine efficacy.We constructed a recombinant vaccinia expressing firefly luciferase and HIV-1 Gag fusion protein based on Tiantan strain which is an attenuated but replication-competent poxvirus(rTTV-lucgag).Using luciferase activity as read out,we firstly analyzed the biodistribution of Tiantan strain poxvirus in mice challenged intraperitoneally.The results demonstrated that this rTTV-lucgag had vigorously replicated in the ovary,uterus and cervix of mice and peaked on days 2 after challenge with the values of 242 273±131 222 RLU/mg protein for ovary,192 613±328 398 RLU/mg protein for uterus and 78 182±13 5049 RLU/mg protein for cervix,on day 5 after challenge,the activity decreased to 66 621±83 630 RLU/mg protein in the ovary which is higher than the activities in any other tissues.Based on above results,the ovary was selected as sensitive target tissues for the subsequent protective evaluation of Gag vaccines;Furthermore,the luciferase activities are tightly correlated with viral titer(r~2=0.7143,p＜0.0001) and Gag expression(r~2=0.5699,p=0.03) in the ovary respectively.Subsequently,we immunized mice with a DNA vaccine expressing HIV-1 Gag immunogen,and then challenged with rTTV-lucgag,3 days after challenge,luciferase activities in the ovaries were determined in DNA vaccine immunized mice as a level of 1 538±462.5 RLU/mg protein which was only a quarter of the level in control mice ovaries(6 006±3 141 RLU/mg protein).These results demonstrated that mice vaccinee had better capacity to suppress the viral replication than control mice,proved that our rTTV-lucgag/mice model had the potential to evaluate protective efficacy of HIV gag containing vaccines.Overall,we concluded that luciferase activity in ovary represents viral replication in vivo and this rTTV-lucgag/mice model can assess protective efficacy of cytotoxic T cell response to HIV gag with less labor-consuming and more sensitivity.Another challenge is the development of safe and efficient vaccine vectors. Currently,viral vectors are the most efficient vectors used for gene delivery that depend on virus physiological binding to host cells and deliver their genetic material into host cells as part of their life cycle.However,several considerations,such as safety concern,high immunogenicity against viral vector itself,limited size for inserts and narrowed tropism for host cells hinder the extensive use of viral vectors in gene therapy and vaccination.DNA vaccine has highly limited vector-related immunogenicity and can be used for multiple times, but its low levels of transfection and expression of the inserted gene in vivo limit its capacity of inducing strong immune responses.Therefore,a safe and efficient delivery system is desirable for gene therapy and vaccine development.Cation polymer Poly[2-(N,N-dimethylamino) ethyl methacrylate](PDMAEMA),like polyethylenimine(PEI),can bind to negative charged DNA through electrostatic interaction,and thus improve the transfection efficiency in vitro,but the high cytotoxicity limits its application in vivo.In studyⅡ,we aimed to develop an efficiency DNA vaccine delivery vector.We PEGylated the high cytotoxic cation polymer PDMAEMA first. Agarose gel electrophoresis,particle size detection and transmission electron microscopy(TEM) results suggested that PEGylated PDMAEMA retained its binding capacity to DNA,and could form a particle with an average diameter at～150nm.Then we incubated the complex with 293T cells for evaluating the cytotoxicity and tranfection efficiency in vitro,and the data showed that when N/P ratio was above than 30,the cytoxicity of PEGylated PDMAEMA was significantly lower than its unmodified form and the difference was further expanded with the increase of N/P ratios.We then analyzed the effect on transfection efficiency of PEGylated polymers using green fluorescent protein (GFP) as reporter and observed that the transfection efficiency of PEGylated PDMAEMA had dramatically decreased and was about one twentieth of the PEI-mediated transfection efficacy.Furthermore,we intranasally primed mice with PEGylated PDMAEMA complexed with plamid DNA expressing HIV-1 CN54 Gag immunogen and then intramuscularly boosted with recombinant Tiantan vaccinia(rTTV) expressing the identical immunogen,we proved that PEGylated PDMAEMA elicited considerable HIV-1 Gag specific T-cell immune response(163±35 SFCs/1×10~6 splenocytes),and this was significantly more than naked DNA(56±5 SFCs /1×10~6 splenocytes,p=0.0052) or PEI complexed DNA(99±14 SFCs/1×10~6 splenocytes,p=0.0441) as intranasal priming.In addition,PEGylated PDMAEMA elicited higher levels of HIV-1 Gag specific antibody responses than naked DNA(p=0.0341).Finally,we demonstrated that PEGylated PDMAEMA had immunostimulatory activity which can induce TNF-αand IL-10 production in murine macrophages,which is independent of the usage of DNA.In summary,PEGylated PDMAEMA reduced the cytotoxicity of PDMAEMA,and was capable of enhancing DNA vaccine mucosal priming effects;Importantly,transfection efficacy in vitro does not correlate to the immunogenicity in vivo for polymer vectors,the adjuvant effects of the vector may enhance the immunogenicity through an adjuvant mechanism.