| Tuberculosis (TB), caused by Mycobacterium tuberculosis, represents one of the most significant health problems in the world. The epidemic has a major negative impact on social and economical development in many poor countries. Most recently, fluid population increasing, the human immunodeficiency virus(H/V) and Mycobacterium tuberculosis(Mtb) dual infections, and multiple-drug resistance strains of Mtb emerging have caused the number of new tuberculosis (TB) cases to explode worldwide. Currently, Mtb has infected approximately two billion individuals in total. TB remains one of the great killers, causing between 2 and 3 million deaths, and an estimated more than 8 million new infections a year. TB is plaguing China that accounted for 15% of incident cases globally. Obviously, reduction of incidence of active TB should target on reduction of the rate of transmission of the disease. There are two main strategies for reducing the transmission rate: (1) rapid diagnosis and promptly treatment of active TB; (2)preventing TB occurrence with an effective vaccine. These two strategies complement each other, but they are not as effective in practical life as needed to combat the disease. Active TB should be detected as early as possible to avoid transmission. Diagnosis and treatment, although effective, demand substantial resources to be properly effected and are now facing considerable hurdles as multi- drug-resistant TB is evolving at an alarming rate in many areas. Prevention of TB by BCG vaccination has not given the expected results despite BCG being one of the most widely used vaccines in the world. Development of new and improved vaccines against TB is therefore an urgent need.As a major secreting protein of Mtb, Ag85A has attracted tremendous interest because of its function on inducing cell immune responses as well as humoral immune reaction. Due to its ability to infect various types of cells and the capability to mediate long-term gene expression, recombinant adeno-associated virus type 2 (rAAV-2) has attracted tremendous interest as a promising vector for gene delivery. To develop new vaccine against Mtb, we have constructed rAAV-2 containing Ag85A gene of mycobacterium tuberculosis (rAAV-2- Ag85A ) and investigated its immunogenicity. Our study aim at providing original clue to preventing Mtb infection and curing active TB, and analyzing the possibilities that introduce rAAV-2-Ag85A into remedial and/or prophylactic vector live vaccine.Ag85A gene was amplified by polymerase chain reaction (PCR) from genome of mycobacterium tuberculosis H37Rv strain, and was cloned into the rAAV-2 expressing vector; pSNAV. The recombinant pSNAV-Ag85A was transfected into BHK-21 cell by using Lipofecta- mineTM 2000. Screening with G418, we isolated a mixed cell line expressing Ag85A (BHK-Ag85A). Then the mixed cell line was infected with helper virus which has function of packaging the rAAV-2. After purification, rAAV-2-Ag85A was obtained. HPLC was used to monitor the purity of rAAV-2-Ag85A. The blot hybridization was used to determine the physical titers of rAAV-2-Ag85A. Expression of rAAV- 2-Ag85A in BHK-21was detected by Western blot. The result of sequencing showed no changes had found in our amplified fragment compared with Ag85A sequence reported in GenBank(AY732095). The results of HPLC showed that the purity of the final rAAV-2 product were higher than 99%. The physical titers of rAAV-2-Ag85A was 2x1012 virus particles/ml(vp/ml). Detected by Western blot, rAAV-2-Ag85A could express one polypeptide with the molecular mass of 32 kD in vitro. These results indicate that the recombinant virus for expressing Ag85A has been successfully constructed. The recombinant virus has high titer and purity and can transduce cultured cells efficiently.To our knowledge, it is the first report that rAAV-2 expressing antigen gene was introduced into Mtb vaccine. To observe the transduction efficiency of rAAV-2-Ag85A in vivo, Balb/C mice were administrated with 5×1011 virus particles/ml (vp/ml) rAAV-2-Ag85A via intranasal route(in). After 3 weeks, cDNA fragment of Ag85A in lung tissue was amplified by RT-PCR and the expression of Ag85A in lung tissue was detected by Western blot. After Balb/C mice were immunized with rAAV-2-Ag 85A via intramuscular injection (im) and intranasal respectively, the contents of Mouse interferon-gamma (mIFN-γ)and Anti-Ag85A antibodies in sera of Balb/C mice were assayed by enzyme-linked immunosorbent assay(ELISA) and Cytotoxic T lymphocyte (CTL) assays were performed with 51Cr release assay. RT-PCR result showed that an about 1Kb fragment had successfully been amplified from mice lung tissue. The expression of exogenous Ag85A in lung tissue had been detected by western blot. Both via intramuscular injection and intranasal route, Balb/c mice were induced to generate antigenic specificity antibody and cytolytic T lymphocyte (CTL), and Th I cytokine, mlFN-γat 10th day after booster dose of rAAV-2-Ag85A. Between via im and in route, there were no significant differences in antibody titers, CTL activities and mIFN-γcontents at majority time phase points. It is indicated that exogenous Ag85A gene can be integrated into the chromosomal DNA of cells in lung tissue and the integrated gene can transcribe and translate effectively. Immunization with rAAV-2-Ag85A can induce both humoral and cellular immune response, as well as mIFN-γaugmentation. Our research has demonstrated that rAAV-2-Ag85A has not only prophylaxis effect but also immunotherapy function because of its capable of enhancement Th I immune response. Maybe rAAV-2-Ag85A has potential value as a therapeutic vaccine for TB. Since immunization with rAAV-2-Ag85A via intramuscular injection and intranasal route has almost the same effect, rAAV-2-Ag85A can be used as a promising candidate for mucosa immunization. In a word, rAAV-2-Ag85A has important value to Mtb infection preventing as a priming vaccine or a priming-booster vaccine, and to TB treatment as a therapeutic vaccine.
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