Prepare Of Lyophilized Recombinant Virus-Based HIV-1 Vaccine And Prime-Boost Strategy

The spread of HIV-1 infection has already seriously threatened humanhealth and economic development. Chemotherapeutic has been limited tocost and the strict nature of the administration regimens, and high mutationrate in virus. These restrictions support that there is a need for thedevelopment of effective vaccine. From the historical experience and longterm consideration, the development safe and effective vaccine is theprevention and control AIDS basic way and one of effective actions.Onlyby using vaccine can control HIV-1 spread.More and more evidence show that cytotoxic T lymphocyte (CTL)responses play an important role in the protection against HIV viruses.Several groups have observed that in acute cases of HIV-1 infection, viralclearance was associated with specific CTL activity. HIV-1 specific CTLswere also found in a number of chronically exposed prostitutes in Gambiawho continue to resist infection with HIV-1.Long-term non-progressorHIV-infected individuals, chronic low virus load and is related with highfrequency CTL.Cytotoxic T lymphocytes (CTL) not only recognizeprocessed viral fragments presented on the infected cell surface and destroyvirus-infected cells, but also target all viral gene products the are expressedduring viral replication. Targeting viral proteins through the development ofspecific CTL responses could aid in lowering the viral load by interferingwith viral assembly.Some type vaccines can elicit cellular immune response, such as DNAvaccine and recombinant virus vaccine .Recombinant non-replicated virus-based vaccinevirus-based vaccine has been widespread applicated in AIDS vaccine.The viral carrier may divide into the replicated and non-replicated viruscarrier. But stems from the safe consideration generally, non-replicatedcarrier has been mostly utilized,such as smallpox virus carrier MVA,adenovirus carrier (Ad5) and so on.rMVA: The smallpox vaccine virus carrier can induce the humoral andcellular immunity and mucous membrane immune response.Therefore ithas been widely utilized in the vaccine research including pre-clinical aswell as in the human experiment of the HIV-1 vaccine . At present, MVA asthe vaccine carrier is selected by many vaccines research team. At presentMVA widely used in the human gene therapy and vaccine research,moreover the animal experiment also proved that recombinant MVA-basedvaccine can induce good immunity protection response.Ad5: The duplication-defected adenovirus lacks the E1 and E3 gene.Itonly duplicate in the specific packing cell line expressing the E1 gene .The duplication-defected adenovirus as the vaccine carrier has alreadyconfirmed that can produce strong humoral immune and CTL response inthe mouse and in the non-human primate, Also can initiate the macaque andchimpanzee's systematic and mucous membrane immunity after the nasaladministration.Construct of hiv recombinant virus-based vaccineIn China, the HIV-1 virus's main popular hypotype is B/Creorganization hypotype. It is reported that 50% of new infected-personin China is result by HIV -1 B/ C hypotype. This article construct thestructure gene (GagPol and Env) of Chinese HIV-1 popular strains intothe viral carrier . By identification in the nucleic acid level and in theprotein level,We obtained recombinant MVA virus-based vaccine andrecombinant adenovirus-based vaccine in view of main HIV-1 popularstrain in China.Lyophilized MVA -based HIV vaccine and produce technicsMVA is sensitive to freeze-thaw cycle repeatedly and easy to losealive.Its thermostability is relatively bad. In order to make the activity ofMVA vaccine remain stable during manufacturing,storage andadministration, through screening , the right protectant compositions andproduce technics had obtained for the first time. The final moisturecontent of freezing-dried recombinant MVA-HIV vaccine was lower than3%. It reconstituted quickly and showed regular physical appearance andstabile potency. In vivo,mice were divided randomly into the liquidvaccination group,the lyophilized vaccination group,and the control group.The mice which immunized with a dose of 107pfu MVA-HIV vaccine whichis stable after freezing-dried produced indistinguishable antibody titerand CTL level with liquid vaccination group(P>0.05). This indicatedthat the lyophilized vaccine indistinguishable with liquid vaccine oncellular immunity and humoral immunity . These results demonstrated thatlyophilized MVA vaccine is safe and high immunogenicity in mice. Inaddition,it can be shipped and stored at the room temperature ,supporting its further evaluation and application in human clinical studies,especially in the remote area.Lyophilized Ad-based HIV vaccine and produce technicsTo prepare the stabilizer of lyophilized Ad-based vaccine,add thestabilizers prepared with various formula into Ad-based vaccine,thenlyophilize the vaccine under appropriate condition. Screen the optimalformula of stabilizer according to the appearance, virus titer andthermostability test of the lyophilized vaccine. Virus titration wascarried out to determine the infectivity of the lyophilized Ad-basedvaccine after exposure at 4, 37 for different time intervals. Theinfectivity titres thus obtained were subjected to regressionanalysis.the half-life (time required for loss of half the original titre,i.e. 0.30 log10 TCID50 based on the degradation constant) oflyophilized vaccine was more than that of liquid vaccine.Thestabilizer consisting of human serum albumin,fucose, mannitol,dextran,sucrose provided good protective effect and suitable for thelarge-scale production for lyophilized Ad-based vaccine.In vivo,mice were divided randomly into the liquid vaccination group,the lyophilized vaccination group,and the control group. The mice whichimmunized with a dose of 108 pfu Ad-based vaccine which is stable afterfreezing-dried produced indistinguishable antibody titer and ELISPOTlevel with liquid vaccination group(P>0.05). This indicated that thelyophilized vaccine indistinguishable with liquid vaccine on cellularimmunity and humoral immunity . These results demonstrated thatlyophilized MVA vaccine is safe and high immunogenicity in mice. Inaddition,it can be shipped and stored at the room temperature ,supporting its further evaluation and application in human clinical studies,especially in the remote area.The strengthen immunity procedure of different dosage-formvirus-based vaccine influence on AIDS vaccine immunityThe powerful cytotoxic T cell effect induced is essential for HIV-1vaccine. This research constructed recombinant hiv Ad-based vaccine andrecombinant hiv MVA-based vaccine. Lyophilized virus-based hiv vaccineand produce technics were obtained for the first time.This experiment usedthe lyophilized recombinant hiv Ad-based vaccine at the beginning of vaccineto exempt and the Prime-boost immunity strategy which the MVA-basedvaccine strengthened. We studied the different dosage-form virus-basedvaccine and different immunity procedure influence on the HIV-1 immunity.The result showed lyophilized hiv Ad-based vaccine prime two times andlyophilized hiv MVA-based vaccine boost can acquire better immunityeffect. This research increased new immunity strategy, provided theorybasis for the next AIDS vaccine immunity procedure improvement, also haslaid the foundation for the future in the non-human primates modelexperiment.