Study On Liposomal Rabies Vaccine For Human Use, Freeze-dried

Rabies is a zoonotic disease caused by the rabies virus. Once infection is established, the mortality rate is 100%. To date, rabies has not been effectively controlled and results in an estimated 55,000 human deaths annually worldwide. The only effective method to prevent rabies is an immediate inoculation of human rabies vaccine. Originally, the first vaccines administered to prevent rabies were nerve tissue derived vaccine (NTV), these vaccines are recognized as poorly immunogenic and highly reactogenic. Today cell culture rabies vaccines that are highly immunogenic and less reactogenic than NTV, such as human diploid cell vaccine (HDCV), purified chick embryo cell rabies vaccine (PCECV), purifed vero cell rabies vaccine (PVRV), and puried duck embryo cell vaccine (PDEV) are available. Routinely the cell-culture vaccines are formulated with inactivated rabies viruses with or without aluminum salt, compared to nerve-tissue vaccines, the cell culture vaccines are generally considered safe and effective at inducing RVNAb. Typically, immunization with human rabies vaccine is divided into a pre-exposure and a post-exposure prophylaxis. The schedules are different according to the recommendations made by individual countries. At present, the intradermal and intramuscular pre-exposure prophylaxis recommended by the WHO involves the repeated injections on days 0,7,21 or 28. The post-exposure prophylaxis recommended by the WHO that includes the Essen regimen, Zagreb schedule, Thai Red Cross schedule and Oxford regimen. These treatments are all laborious and time consuming. The Essen regimen involving the repeated injections (2.5IU/dose) on days 0, 3,7,14 and 28, is the "gold standard" for all PEP regimens.The rabies vaccine for human use has also experienced the 21 injections, 14 injections, and 7 injections to the current 5 injections. The purpose of this paper is to prepare the high potency liposomal rabies vaccine, to reduce inoculations to 2 injections for pre-exposure prophylaxis, and reduce inoculations to 3 injections for post-exposure prophylaxis, and shorten the time for full immunization.In this study, we used liposomes as an adjuvant for rabies vaccine in order to make freeze-dried liposomal rabies vaccine with high potency for human use, we explore its pharmaceutical and toxicological properties, observe the prepared vaccine for morphology under electron microscope, and determine for envelope rate and release in vitro. Analyze the particle distribution and size of the vaccine by laser diffraction particle size analyzer; perform local stimulation, hypersensitivity and abnormal toxicity tests on the prepared vaccine. The prepared rabies virus liposome vaccine under electron microscope were in forms of round or elliptic particles distributed evenly, at a mean size of 12.25μm. The envelope rate of the vaccine was 60%-70%. The vaccine showed no stimulation or abnormal toxicity and was qualified in hyper sensitivity test.The potency of the vaccine was tested according to the NIH protocol. The data indicated that liposome could enhance the rabies vaccine to induce effective protection in mice against rabies virus. At the same amount rabies antigen, the potency of the RabV is 3.1 IU/dose,3.4IU/dose, 3.2IU/dose; the potency of LipoRabV is 6.1 IU/dose, 7.2 IU/dose, 6.0 IU/dose. Mice immunized by single IP or 5 injections with RabV or LipoRabV. The RVNA in the sera of immunized mice was detected using RFFIT method. p<0.05. The BALB/c mice were immunized with PBS (control) or RabV or LipoRabV vaccines, three days after immunization, the levels of IL-2 or IFN-γin the sera were detected using ELISA assays. p<0.05,for IL-2; p<0.01, for IFN-γ.For NK cell activity assay, the up-regulation of NK was expressed as the percentage of FITC-negative and PE-positive cells in lymphocyte population. The results indicated that LipoRabV produced the same effective NK cells as RabV in the BALB/c mice. P>0.05.Liposome could facilitate the inactivated rabies vaccine (RabV) to induce more vigorous production of rabies virus neutralizing antibody (RVNA) in BALB/c mice and beagles. We established preliminary pre- and post-exposure prophylaxis schedules for LipoRabV. LipoRabV (0/14) could elicit similar RVNA level and achieve the same survival rate as RabV (0/7/28) by pre-exposure prophylaxis schedules in mice and beagles. LipoRabV (0/3/14) could elicit similar RVNA level vs. RabV (0/3/7/14/28). The data indicated that the three-shot liposome-enhanced rabies vaccine could achieve a higher protection rate (survival rate 56.2%) by post-exposure prophylaxis than the RabV group (survival rate 40.6%) in mice. The data also indicated that the three-inoculation liposome-enhanced rabies vaccine could achieve the survival rate (80.0%) vs. RabV (70.0%) by post-exposure prophylaxis in beagles. The results showed that the immunization schedule for LipoRabV could be preliminarily determined at days 0, 14 for pre-exposure prophylaxis and at days 0,3, 14 for post-exposure prophylaxis.Here, we investigated the effect of a three-shot rabies vaccine with liposome adjuvant on BALB/c mice, and found that this inactivated rabies vaccine with liposome (LipoRabV) could facilitate the inactivated rabies vaccine (RabV) to induce an earlier and more vigorous production of rabies virus neutralizing antibody (RVNA), resulting in more effective protection of BALB/c mice from the rabies virus challenge. More importantly, this LipoRabV procedure could finish rabies PEP in half a month as compared to the 28 days requirement for Essen regimens. We conducted some preliminary studies on the cellular and humoral immunity of human freeze-dried liposomal rabies vaccine. We found that the liposome could enhance the cellular and humoral immune response to rabies vaccine. This could be the mechamsm of increased protective potency of the vaccines. We completed in vivo studies for immunization schedules and established an initial immunization schedule for human freeze-dried liposomal rabies vaccine. Compared with the schedules mentioned above, our schedule has the advantages of fewer injections and a shorter immunization period.