Construction Of A DNA Vaccine By Linkage Of Mycobacterium Tuberculosis Ag85B Gene With Protein Transduction VP22 Gene And Its Protective Effect Evaluated In Mice

TB is an ancient infectious disease that remains a major threat to public health worldwide. The morbidity and mortality of TB has resurged in recent years in many places around the world. Although BCG vaccination has been widely used to prevent TB worldwide, it has shown considerably variable efficacy with insufficient protection against pulmonary tuberculosis in adults. Besides, BCG itself has some formidable drawbacks. Therefore, the novel more efficient and safer vaccines and/or vaccination strategies are urgently needed.DNA vaccines have attracted intensive attentions among the new generation of TB vaccines development due to its unique properties. To date, more than thirty DNA vaccines encoding mycobacterial immunodominat antigens have been tested in preclinical animal models with beneficial effects. Unfortunately, their performance generally fails to surpass the BCG, especially in large animals and primates. However, the recent licensure of four DNA vaccines for veterinary use including larger mammals (e.g. horses and pigs) and good performance of DNA vaccines against cancer, AIDS and malaria in clinical trials highlighten the potentiation of this technology. Therefore increasing the potency of DNA vaccines has been the priority of the research.Among the optimization strategies of DNA vaccines, the application of protein transduction technology is a potential and promising approach. To date, it is well documented that the tegument protein VP22 of some alphaherpesvirus, such as herpes simplex virus 1 (HSV-1), bovine herpesvirus 1 (BHV-1), Marek's disease virus 1 (MDV-1), and pseudorabies virus (PRV), has an unique intercellular trafficking property, and is capable of transporting from the transfected cell to surrounding bystand cells where the proteins were taken up through a nonclassical undefined mechanism. Furthermore, VP22 can ferry genetically fused foreign proteins, peptides and nuclear acids to cells in vitro and in vivo without loss of biological functions of the cargoes. Many experiments have shown that these VP22 molecules have the potential to act as immunoadjuvants to enhance the potency of conventional DNA vaccines, self-replicating RNA vaccines, suicidal DNA vaccines, recombinant virus vector vaccines and gene therapeutic efficacy of the recombinant adenovirus-based vaccines as well as the DNA-based vaccines. It is known that bovine herpesvirus-1 VP22 (BVP22) is the most efficient natural protein transduction molecules reported so far. It is unclear whether or not BVP22 can be used to enhance the efficacy of TB DNA vaccines. To our knowledge, however, there have been no reports with respect to the application of protein transduction to TB DNA vaccines and a research on this question will widen the application of protein transduction technique, and provide a promising approach for enhancing the potency of TB DNA vaccines.Antigen 85B is one of the major immunodominant proteins during the MTB infection. Researches with Ag85B have showed that the plasmids encoding Ag85B alone or Ag85B in combination with other immunodominant proteins could induce potent cellular and humoral immune response, significantly reduce the bacterial loads in spleen and lungs and prolong the survival time of infected animals (mice and gninea pigs) after immunization. Furthermore, researchers have reported that expressing Ag85B DNA vaccination has immunotherapeutic effects in addition to prophylactic efficacy. Therefore we chose these genes encoding mycobacterial Ag85B and BVP22 as our genes of interest to construct the co-expression DNA vaccine and evaluated the influence of BVP22 on immunogenicity and protection of TB DNAAg85B vaccine against virulent Mycobacterium tuberculosis H37Rv challenge in C57BL/6 mice. The experiment was divided into two parts.Part A Construction of a DNA vaccine by linkage of Mycobacterium tuberculosis Ag85B and Bovine herpesvirus 1 VP22 and its immunogenicity evaluation in C57BL/6 miceObjetive To construct the DNA vaccine encoding the fusion protein of Mtb Ag85B with BVP22 and to evaluate the immunological properties of the vaccine in C57BL/6 mice in comparison with DNA expressed Ag85B alone.Methods 1. Construct DNA vaccines containing fusion protein BVP22-Ag85B, Ag85B and BVP22 in eukaryotic expression vector pcDNA3.1(+) using molecular biological technique. Following verification with DNA sequencing and restrict endonuclease digestion, the purified constructed plasmids were transfected into HeLa cells line with LipofectAMINETM 2000 and expression of Ag85B and BVP22-Ag85B in HeLa cells were determined by RT-RCR and Westernblotting.2. Specific-pathogen free grade, body weight 16-18 g, aged 4-6 weeks female C57BL/6 mice were randomly assigned to 6 groups and immunized intramuscularly with the purified plasmid constructs for three times at 2-week intervals. At the same time, mice vaccinated subcutaneously once with BCG (106CFU per animal) at the time of the first prime dose of candidante DNA vaccine and mice immunized intramuscularly with PBS, empty vector and plasmid containg BVP22 only for three times at 2-week intervals were designated as positive control and negative controls. Six weeks after the last immunization, five mice from each group were sacrificed to determine the immunogenicity of the vaccines. The total specific antibody titres were determined by indirect ELISA, the specific spleen lymphocyte proliferation responses were measured by MTT colorimetry and the numbers of IFN-y producing and IL-4 producing lymphocytes in the spleen were quantified by ELISPOT.Results 1. Plasmids have been constructed correctly by DNA sequencing and restrict endonuclease digestion, and the corresponding mRNA and protein were detected in the transfected eukaryotic cells.2. Immunization of both plasmids of pcAg85B and pcBVP22-Ag85B can induce potent cellular and humoral immune responses in C57BL/6 in comparison with the negative controls. Compared with pcAg85B, pcBVP22-Ag85B immunization induced higher level of cellular and humoral immunity in mice, indicated as significantly higher antibody titre and stimulated index as well as more number of IFN-y producing lymphocytes in the spleen, which was slightly lower than that of BCG vaccination but the difference was no statistically significant. No significant difference of IL-4 producing lymphocytes numbers in the spleen were detected in all groups.Conclusion Plasmid encoding fusion protein of mycobacterial Ag85B and BVP22 can induce a more efficient cellular and humoral immune response in C57BL/6 compared with DNA expressing Ag85B alone. We presumed that this observed enhancement of immune response in mice might be related with improved immunogenicity of the DNA vaccine due to the addition of BVP22 to mycobacterial Ag85B, which enhanced the capacity of antigen into different cells and subsequent presentation. Our presumption requires further study. Part B Protective effect of co-expressing mycobacterial Ag85B and BVP22 plasmid evaluated in mice infected with Mycobacterium tuberculosis H37RvObjective In order to evaluate whether an improved immunogenicity of a DNA vaccine was accompanied with a better protection against mycobacterial infection, we further invested the protective effect of plasmids mentioned above in mice infected intravenously with Mycobacterium tuberculosis H37Rv.Methods Specific-pathogen free grade, body weight 16-18g, aged 4-6 weeks female C57BL/6 mice were randomly assigned to 6 groups. Six weeks after the completion of gene immunization, immunized mice were challenged with 8×106 CFU Mycobacterium tuberculosis H37Rv via lateral tail vein in the animal biosafety level-3 laboratory. All mice were sacrificed at day 35 post challenge. The lungs and spleens were removed respectively, and the numbers of CFU in lungs and spleens as well as the histopathologic changes in lungs were determined. The pathological sections of lungs were stained with both acid-fast staining and haematoxylin and eosin (H&E) staining. Besides, the general survival conditions, the time of half death (T50) and survival rate of infected mice in 35 days were observed. At the same time, splenocytes from two mice in each group as well as two normal uninfected mice were prepared and the percentage of CD4+CD25+T cells in the splenocytes were detected by flowcytometry.Results There were no significant difference observed in all experimental groups at the first two weeks in view of body weight and general survival condition. The body weights in the mice of PBS, empty vector and pcBVP22 controls began to decline from the third week postchallenge. Death began at day 17 in PBS control, day 19 in empty vector control and day 18 in pcBVP22 control postchallenge, respectively. As for pcAg85B and pcBVP22-Ag85B immunized mice, death began at the day 24 and day 30 postchallenge, respectively. At the day 35 postechallenge, there are 2 mice survived in PBS control,2 mice survived in empty vector control,3 mice survived in pcBVP22 control,6 mice survived in pcAg85B immunized group and 9 mice survived in pcBVP22-Ag85B immunized group, while no death happened in BCG vaccination group. The times of half death (T50) in pcAg85B and pcBVP22-Ag85B immunization groups were even more than one week longer in comparison with that of PBS, empty vector and pcBVP22 controls, respectively, and T50 of pcBVP22-Ag85B immunization group was more than one week longer compared with pcAg85B immunized mice. Results of the CFU counts in the lung and spleen tissues of mice showed that significant reduction of pulmonary and splenic bacterial loads were detected in pcAg85B (5.3174±0.0276/lung and 5.3328±0.0774/spleen) and pcBVP22-Ag85B (4.9313±0.0592/lung and 5.0031±0.1031/spleen) immunized mice in comparison with the PBS (5.9443±0.0345/lung and 6.0065±0.0482/spleen), empty vector (5.9362±0.0192/lung and 6.0039±0.0191/spleen) and pcBVP22 controls (5.9416±0.0312/lung and 5.9794±0.0559/spleen), respectively (P<0.05). Although there are slightly more CFU counts in the lungs and spleens of pcBVP22-Ag85B immunizated mice than that of BCG vaccination (4.7813±0.1129/lung and 4.8669±0.1252), no significantly statistical difference were found between them (P>0.05). Results of pathological changes in the lungs showed that there were extensive hyperaemia, infiltrate, necrosis, consolidation and multiple coalescing granulomatous lesions were found in PBS, empty vector and pcBVP22 controls. The basic structure of pulmonary alveoli disappeared in these three groups. In contrast, pcAg85B and pcBVP22-Ag85B immunized mice showed obviously improved lung pathology with dramatically suppressed granulomatous lesions and inflammations, while BCG vaccinated mice had basically normal structures of pulmonary alveoli and slight inflammations. Results of acid-fast stainging were consistent with the HE staining in general. Replication of Mycobacterium tuberculosis in the lung and spleen tissues was significantly inhibited in pcAg85B, pcBVP22-Ag85B and BCG immunized mice, with the hierarchy of BCG, followed by pcBVP22-Ag85B and then pcAg85B immunization. However, there were much higher density of acid-fast bacilli distributed in the lungs of mice immunized with PBS, empty vector and pcBVP22. Results of flow cytometry showed that there are significant higher percentage of CD4+CD25+T cells in the spleens of PBS control mice after infection with virulent Mycobacterium tuberculosis H37Rv compared with that of the normal uninfected mice (14.2% vs 0.4%). pcAg85B, pcBVP22-Ag85B and BCG immunization led to prominent reduction of the percentage of CD4+CD25+T cells in the spleens of mice in comparison with that of PBS control. Compared with pcAg85B immunization, there was much lower percentage of CD4+CD25+T cells in the spleens of mice immunized with pcBVP22-Ag85B (1.2%vs 0.9%), which is slightly higer than BCG group (0.6%).Conclusions Compared with PBS, empty vector and pcBVP22 controls, both plasmids encoding either fusion protein of mycobacterial Ag85B and BVP22 or Ag85B alone induced efficient protection against virulent Mycobacterium tuberculosis H37Rv challenge in C57BL/6, indicated as significant inhibition of mycobacterial growth in the lungs and the spleens, obviously improved lung histopathologies and much longer survival times. Coexpressed pcBVP22-Ag85B elicited a better performance in comparison with pcAg85B. Besides, compared with PBS control, both plasmids (pcAg85B and pcBVP22-Ag85B) and BCG inoculations decreased obviously the percentage of CD4+CD25+T cells in the spleens of infected mice. We supposed that the better protection in coexpressing plasmid group might be related with capacity of BVP22 to enhance the immunogenicity of DNA vaccine. More studies is required to identify whether this good performance is also related with the further decrease in the percentage of CD4+CD25+T cells in the spleens of infected mice.