A Novel HIV DNA Vaccine Based On Salmonella Typhi Ty21a Bacterial Ghosts

Acquired immunodeficiency syndrome (AIDS) is a life-threatening viral disease. HIV infection has become a serious public health problem, thus further prevention and control of the HIV infection is an urgent and important task worldwide. After more than 20 years of arduous research, significant progress has been made in anti-HIV drugs. Although no desired success was achieved, there has been encouraging progress in the development of vaccines against HIV infection. The large-scale trial of a potential AIDS vaccine in Thailand in 2009 has indicated that AIDS vaccines may reduce the risk of HIV infection by more than 31%, suggesting that a safe and effective HIV vaccine is still an important strategy to control HIV infection. Current studies have shown that DNA vaccine has the great potential to be developed into an ideal vaccine against HIV infection, because it may effectively induce cellular, humoral and mucosal immune responses, but its low immunogenicity may limit further development. Priming with DNA vaccines and boosting with other vaccine formulations will be an important strategy for HIV vaccine development. In this study, Salmonella typhi Ty21a bacterial ghosts were used as a delivery vector of DNA vaccine, while HIV Env gp140 was used as a pattern antigen, constructing a novel bacterial ghosts based HIV DNA vaccine. This vaccine was evaluated using different vaccination pathways in BALB/c mice. Further, Ty21aBG (DNA) vaccine prime—recombinant protein vaccine or recombinant live viral vaccine boost as immunization strategy was evaluated also..1. Production and identification of Salmonella typhi Ty21a bacterial ghosts(1) Electroporation of Salmonella typhi Ty21a: An electroporation method was used in this study to transform pMBE plasmid expressing protein E of bacteriphage PhiX174 into Salmonella typhi Ty21a by optimizing transformation conditions with selected electronic voltages. The results showed that high voltage (1 KV) cause cell death, while plasmid can be successfully transformed at voltage 250 V or 500 V, with the efficiency of the latter slightly higher than that of the former. Thus, bacteria Ty21a can be efficiently transformed by using optimal electroporation parameters with voltage at 500V, electric resistance at 200Ω, and capacitance at 25μF.(2) Production of Salmonella typhi Ty21a bacterial ghosts: Bacterial ghosts were produced by controlled expression of PhiX174 lysis protein E in the Ty21a. Recombinant bacteria Ty21a (pMBE) were grown at 28℃until reaching OD600 of 0.3~0.4. By the time, the bacterial cultures were divided into two batches: temperature of one was up shifted from 28℃to 42℃to induce the expression of gene E, while the other was still cultured at 28℃. Compared with bacteria Ty21a, recombinant bacteria Ty21a (pMBE) lysis occurred 1 h after temperature shifting and completed 4h after induction. The lysis rate of ghost induction in non-lyophilized Ty21a was 96.77%.(3) Identification of Salmonella typhi Ty21a bacterial ghosts: The biological characters of Ty21a bacteria ghosts such as morphology was analysed in this section. Results show that generation of ghosts in the recombinant bacteria Ty21a was performed successfully. Ty21aBG show no obviously alterations in cellular morphology compared to unlysed cells except for the lysis tunnel. Diameter of the lysis tunnel ranged from 200nm to 400nm by scanning electron microscopy. Bacterial ghosts maintain structural integrity, but the cell envelope is collapsed comparing to the uninduced bacterial cells because of the lost of most cytoplasmic material. However, no bacterial growth was detected in lyophilized bacterial ghosts because of lysis and freeze-dried treatments, showing good safety.(4) Optimization of loading and transfection conditions of Ty21aBG based on the model plasmid pEGFP-N1: The green fluorescent protein expression plasmid (pEGFP-N1) was used to optimize the loading procedure of Ty21aBG, and murine macrophage cell line RAW264.7 was performed for the in vitro transfection experiments. The results indicated that loading temperature and time had no significant effect on the loading efficiency, while the concentration of plasmid DNA was positively correlated with the load efficiency. On average, milligram of bacterial ghosts could be loaded with a concentration of 6 ug DNA, corresponding to approximately 103 copies of plasmids per bacterial ghost. In vitro transfection experiments revealed that Ty21aBG may efficiently target plasmid pEGFP-N1 to RAW264.7 cells, and thus express green fluorescent protein. Therefore, Ty21a bacterial ghosts can be used as a novel DNA vaccine delivery vector, not only with high loading efficiency, but also effectively target DNA to antigen presenting cells, thus the expression of antigen. 2. Evaluation of Ty21aBG-based HIV DNA vaccine efficacy using different vaccination pathways(1) Preparation of Ty21aBG-based HIV DNA vaccine and in vitro transfection experiments: The Ty21aBG based HIV DNA vaccine was produced by loading HIV DNA plasmid pSV140 into Ty21aBG according to the optimized loading conditions, followed by in vitro transfection experiments. From the experiment results we found that Ty21aBG loaded with pSV140 plasmid can effectively deliver the plasmid into RAW264.7 macrophage, and efficiently express HIV-1gp140 protein.(2) Evaluation of Ty21aBG-based HIV DNA vaccine via subcutaneous vaccination pathway: BALB/c mice were subcutaneously (s.c.) vaccinated with Ty21aBG (DNA) vaccine, and detected for gp120-specific IgG and secretory IgA (sIgA) antibodies as well as cellular immune response at 10 days after the last vaccination. The results indicated that compared with the naked DNA vaccine, Ty21aBG (DNA) vaccine induced significantly higher titers of specific IgG antibody with the titer of IgG1 slightly higher than that of IgG2a, indicating its ability in eliciting a balanced Th1/Th2 immune response, and in increasing immune efficiency of DNA vaccines. In addition, this novel HIV DNA vaccine was able to induce efficient cellular and mucosal immune responses as well. The mechanism of Ty21aBG (DNA) vaccine in increasing immune responses was further carried out based on the in vitro RAW264.7 cells and in vivo BALB/c mouse models. The results suggested that compared with the naked DNA vaccine, the level of IL-10 cytokine secretion significantly increased in both infected RAW264.7 cells and vaccinated BALB/c mice in the early stage of Ty21aBG (DNA) vaccination (P < 0.01), while there was no significant difference on the secretion of IFN-γå'ŒIL-12. These results suggest that Ty21aBG-based DNA vaccine may active Th2 immune responses, and enhance humoral immune responses, maintaining a balance between humoral and cellular immune responses.(3) Evaluation of Ty21aBG-based HIV DNA vaccine via intramuscular vaccination pathway: BALB/c mice were intramuscularly (i.m.) vaccinated with Ty21aBG (DNA) vaccine and HIV-1 gp120-specific IgG antibodies and classes were further evaluated 10 days after the last vaccination. Compared with the naked DNA vaccine, Ty21aBG (DNA) vaccine induced high titers of specific antibodies without any adjuvant. The vaccination also kept a balanced Th1/Th2 immune responses with the titer of IgG1 slightly higher than that of IgG2a, indicating its ability in increasing Th2 immune responses.(4) Evaluation of Ty21aBG-based HIV DNA vaccine via intranasal and intragastric vaccination pathway: In order to evaluate mucosal immune responses potentially induced by the candidate vaccine, BALB/c mice were vaccinated with Ty21aBG (DNA) vaccine intranasally (i.n.) and intragastrically (i.g.), respectively, gp120-specific IgG antibodies and classes were further detected 10 days after the last vaccination. The results demonstrated that compared with the naked DNA vaccine, Ty21aBG (DNA) vaccine induced significantly higher level of antigen-specific antibody immune responses without any adjuvant (P < 0.05), further illustrating that Ty21aBG-based HIV DNA vaccine may enhance immune responses.3. Immune efficacy evaluation of Ty21aBG-based HIV DNA vaccine combined with other formulations of HIV vaccine(1) Ty21aBG (DNA) vaccine prime-recombinant gp120 protein vaccine boost strategy: BALB/c mice were s.c. prime vaccinated with Ty21aBG DNA vaccine, followed by i.m. boost vaccinated with recombinant Ty21aBG (gp120) protein vaccine, and immune responses were detected as above. The results revealed that this prime-boost vaccine strategy was able to induce antigen-specific humoral immune responses without any adjuvant. More importantly, the vaccination elicited both high titers of gp120/ThaiB- and gp120/CRF01_AE-specific IgG antibodies. Compared with Ty21aBG (DNA) vaccine, this prime-boost vaccination strategy induced significantly higher titer of gp120/CRF01_AE-specific IgG titers although the titer of gp120/ThaiB-specific IgG antibodies is similar between the two groups with or without protein boost. Therefore, Ty21aBG (DNA) vaccine prime-recombinant gp120 protein vaccine boost vaccination could elicit more effective humoral immune responses than Ty21aBG (DNA) vaccine alone.(2) Ty21aBG (DNA) vaccine prime-recombinant live virus vaccines boost strategy: BALB/c mice were s.c. immunized with Ty21aBG (DNA) vaccine, followed by i.m. vaccinated with recombinant live virus vaccine (MVTT) and and immune responses were detected as above. The results revealed that this prime-boost vaccine strategy can effectively induce gp120-specific IgG, particularly IgG2a antibodies, indicating the ability of the MVTT virus vaccine in enhancing a Th1-type cellular immune response, maintaining a balance between humoral and cellular immune responses.4. Conclusion: In this study, Salmonella typhi Ty21a bacterial ghosts were successfully produced by controlled expression of lysis protein E of bacteriophage phiX174. The generated Ty21a bacterial ghosts were able to effectively target DNA vaccines to antigen presenting cells, and further express protein antigens, suggesting its potential use as a novel DNA vaccine delivery system. Ty21aBG based HIV DNA vaccine was proven to be effective to induce strong systemic and/or mucosal immune responses in the vaccinated mice via subcutaneous, intramuscular and mucosal pathways in the absence of adjuvants. Furthermore, prime with Ty21aBG (DNA) vaccine and boost with recombinant gp120 protein or live virus vaccine was shown to significantly enhance antigen-specific immune responses in the vaccinated mice. Taken all of the above into full consideration, the developed Ty21aBG (DNA) vaccine could be used as a promising vaccine candidate against HIV infection.