Overexpression and purification of c-terminal fragment of the passenger domain of hap protein from nontypeable haemophilus influenzae in a highly optimized escherichia coli expression system

Nontypeable Haemophilus influenzae [NTHi] is a common cause of respiratory tract disease and initiates infection by colonization in nasopharynx. The Haemophilus influenzae [H. influenzae] Hap adhesin is an auto transporter protein that promotes initial interaction with human epithelial cells. Hap protein contains a 110 kDa internal passenger domain called "HapS" and a 45 kDa Cterminal translocator domain called "Hapbeta". Hap adhesive activity has been recently reported to be connected to its Cell Binding Domain [CBD] which resides within the 311 C-terminal residues of the internal passenger domain of the protein. Furthermore, immunization with this CBD protein has been shown to prevent bacterial nasopharynx colonization in animal models. To provide enough amounts of pure HapS protein for vaccine studies, we sought to develop a highly optimized system to overexpress and purify the protein in large quantities. To this end, pET24alpha-cbd plasmid harboring cbd sequence from NTHi ATCC49766 was constructed and its expression was optimized by testing various expression parameters such as growth media, induction temperature, IPTG inducer concentration, induction stage and duration. SDS-PAGE and Western-blotting were used for protein analysis and confirmation and eventually the expressed protein was easily purified via immobilized metal affinity chromatography [IMAC] using Ni-NTA columns. The highest expression level of target protein was achieved when CBD expressing E. coli BL21 [DE3] cells were grown at 37°C in 2xTY medium with 1.0 mM IPTG at mid-log phase [OD[600 nm] equal to 0.6] for 5 hrs. Amino acid sequence alignment of expressed CBD protein with 3 previously published CBD amino acid sequences were more than%97 identical and antigenicity plot analysis further revealed 9 antigenic domains which appeared to be well conserved among different analyzed CBD sequences. Due to the presence of high similarity among CBD from NTHi ATCC49766 and other NTHi strains, CBD protein expressed here sounds to be theoretically ideal as a universal candidate for being used in vaccine studies against NTHi strains of various geographical areas. Further investigations to corroborate the potency of this protein as a vaccine candidate are under process

endogenous immune adjuvant released by necrotic cells for enhancement of DNA vaccine potency

Improving vaccine potency in the induction of a strong cell-mediated cytotoxicity can enhance the efficacy of vaccines. Necrotic cells and the supernatant of necrotic tumor cells are attractive adjuvants, on account of their ability to recruit antigen-presenting cells to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells. To evaluate the utility of supernatant of necrotic tumor cells as a DNA vaccine adjuvant in a murine model. The supernatant of EL4 necrotic cells was co-administered with a DNA vaccine expressing the glycoprotein B of Herpes simplex virus-1 as an antigen model under the control of Cytomegalovirus promoter. C57BL/6 mice were vaccinated three times at two weeks intervals with glycoprotein B DNA vaccine and supernatant of necrotic EL4 cells. Five days after the last immunization, cell cytotoxicity, IFN-gamma and IL-4 were evaluated. The obtained data showed that the production of IFN-gamma from the splenocytes after antigenic stimulation in the presence of the supernatant of necrotic EL4 cells was significantly higher than the other groups [p<0.002]. The flow cytometry results showed a significant increase in the apoptosis/necrosis of EL4 cells in the mice immunized with DNA vaccine and supernatant of necrotic EL4 cells comparing to the other groups [p<0.001]. The supernatant of necrotic cells contains adjuvant properties that can be considered as a candidate for tumor vaccination

[Designing and construction of bicistronic plasmid pIRESIgk/ mIL18/Fc: potential implications for vaccine investigations]

IL18 is a cytokine that plays an important role in the T-cell-helper type 1 [Th1] response and hence, plasmid-encoded IL18 is considered as a potent genetic adjuvant for DNA vaccine studies. In this study, a bicistronic eukaryotic plasmid capable of secreting a more stable mouse IL18 [fused with Fc gamma 2a fragment] was constructed and expression of this chimer cytokine was also assessed. RNA purified from stimulated mouse spleenocytes and then cDNA corresponding to mouse IL18 [mIL18] and Fc gamma 2a fragments were constructed by RT-PCR. Sequential subcloning of mIL18 and IgG2aFc fragments first into pSL1180 and then pSecTag2 plasmids resulted in the fusion of mIL18/Fc and addition of immunoglobulin kappa signal sequence [Igk/mIL18/Fc], respectively. Final cloning of Igk/mIL18/Fc sequence downstream of CMV promoter into the NheI/XmaI sites of pIRES2-GFP plasmid and led to the construction of pIRES-Igk/mIL18/Fc plasmid, which was transfected to HEK293T cell line by Turbofec Transfection reagent and expression analysis, was evaluated by ELISA assay. Restriction enzyme analysis of pSL-mIL18 pSL-mIL18/Fc pSec-mIL18/Fc and pIRESIgk/ mIL18/Fc plasmids with the enzymes that were applied for clonings led to the isolation of fragments with expected size and then plasmid of pIRES-Igk/mIL18/Fc was also confirmed following sequencing reactions. Moreover, expression and secretion of mIL18 to the medium was evidenced in transfected 293T cells, compared to non-transfected controls. pIRES-Igk/mIL18/Fc plasmid possesses the capacity of the cloning and expression of putative antigen gene under the direction of IRES sequence, and also expression of mIL18 as a great secretive genetic adjuvant. This results can be useful to design an efficient DNA vaccine especially for inducing host cellular immune response, moreover, cab be considered a promising for accessing to new generation of DNA vaccine

Evaluation of Bax encoding plasmid for increasing efficacy of DNA vaccine plasmid encoding gB of Herpes Simplex virus type 1

Evaluation of Bax encoding plasmid for increasing efficacy of DNA vaccine plasmid encoding gB of Herpes Simplex Virus type 1. Materials and Methods: We compared three different dosages of Bax encoding plasmid [pcbax] including 10, 25 and 50 ?g of plasmid DNA. They were co-injected ineradermally with glycoprotein B [gB] of herpes simplex virus [HSV]-1 encoded plasmid [pcgB] in C57BL/6 mice to elicit immune responses to protect against lethal HSV-1 challenge. Immune responses to the antigen were assessed by lymphocyte proliferative responses and cytokine [INF-gamma and IL-4] release assays. The study demonstrates that the mice immunized with 25 micro g pcbax together with pcgB have more efficient protection than the mice immunized with 10 and 50 micro g of pcbax and pcgB. Analysing of cell-mediated responses show that the mice immunized with 25micro g pcbax and pcgB induce stronger lymphocyte proliferative responses and higher levels of INF-gamma and IL-4 compared to the mice are received 10 and 50 micro g of pcbax and pcgB. The data show that co-immunization with 25 micro g of pcbax and pcgB increase immune responses compared to 10 and 50 micro g of pcbax and pcgB. This can be considered a promising approach for development an efficient DNA vaccine against HSV-1 or other pathogens