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This work lays the foundation for developing a single Ad vector encoding …

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- An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes

Plasmids, viruses and cells

Plasmids pVAX1 was from Invitrogen. Plasmids pShuttle-CMV and pAdEasy-1, designed for rAd vector construction, have been described previously [51]. DEN viruses DEN-1 (Nauru Island), DEN-2 (New Guinea C), DEN-2 (H87) and DEN-4 (H241) were gifted by Dr. A. Falconar (University of Oxford, Oxford, UK). Ad5 transformed HEK 293 cell line, Baby Hamster Kidney (BHK 21) cell line, the monkey kidney cell line LLCMK2, the mosquito cell line C6/36, and the 3H5 mAb-producing hybridoma cell line, HB46, were from American Type Culture Collection, Virginia, USA, and were propagated as described earlier [42].

Preparation of DEN virus stocks

DEN virus stocks were prepared by infecting C6/36 cells at low multiplicity for two weeks with media replacement at one week. The resultant infected cell culture supernatant was used as the source of DEN viruses. Viral titers were determined by plaque assay on LLCMK2 cells [42,43].

Construction of rAd-C and rAd-Bg viruses

The design and construction of the bivalent gene, EDIII-4/2, have been described previously [43]. This gene was cloned into plasmid pVAX1 and verified to encode the predicted ~28 kDa bivalent protein in a rabbit reticulocyte expression system (TNT system from Promega). The 3' end of this chimeric gene was fused in-frame with the 5' end of the GFP gene and cloned into the Ad shuttle plasmid pShuttle-CMV. The EDIII-4/2-GFP expression cassette was then inserted into the E1 region of the Ad5 genome of plasmid pAdEasy-1 by in vivo recombination in E. coli BJ5183 [51]. The resultant rAd plasmid was digested with Pac I to eliminate plasmid sequences and transfected into HEK 293 cells to rescue the rAd-Bg virus. The rAd-C virus was constructed in parallel using a similar strategy except that empty pShuttle-CMV plasmid was used during in vivo recombination in E. coli. Recombinant viruses were amplified, purified on CsCl gradients, dialyzed against 1× PBS containing 10% glycerol, sterilized using syringe filters and titrated on HEK 293 cells. Both viruses yielded fairly comparable titers, within 3–4 folds of each other.

Detection of transgene expression in rAd-Bg virus-infected cells

HEK 293 cells were infected at ~25 PFU/cell, for ~24 hours. The expression of the GFP tag was detected by direct fluorescence microscopy. The DEN-2 and DEN-4 EDIII components of the bivalent antigen were detected by IFA, using mAb 3H5 (DEN-2 virus-specific) and MAB8704 (DEN-4 virus-specific), in conjunction with anti-murine IgG-rhodamine conjugate [42]. Expression of the transgene was also detected in infected, [35S]-radiolabeled cells by immunoprecipitation, using the 3H5 mAb as described previously [42].

Mouse immunization

Balb/c mice (4–6 weeks old; 5 animals per group) were immunized using a rAd prime/plasmid boost regimen. The control group was injected intraperitoneally (i.p) with 108 PFU of rAd-C vector (in 200 μl of 1× PBS), followed 15 days later by three intradermal (i.d) doses, again 15 days apart, of 50 μg plasmid pVAX1 vector (in 200 μl of 1× PBS). A similar regimen was followed for the test group, which was primed i.p. with 108 PFU of rAd-Bg vector and boosted i.d. with 50 μg plasmid pVAX-EDIII-4/2 vector. Blood was drawn 1 week after priming and after each boost for seroanalysis. Animal experiments were performed after approval of the Institutional Animal Ethics Committee.


Antibody titers in sera of immunized mice were determined by ELISA, in 96-well plates, using monovalent and bivalent r-EDIII proteins or DEN viruses as capture antigens in conjunction with anti-murine IgG-HRPO secondary antibody conjugate/3, 3' 5, 5' tetramethyl benzidine substrate, as previously described [43]. The presence of anti-DEN-2 and anti-DEN-4 virus antibodies in immune sera (diluted 1:100) was also detected by IFA of DEN-2 and DEN-4 virus-infected BHK cells (0.1 PFU/cell) at 24 hours post-infection. Virus-bound antibodies were detected using anti-mouse IgG-FITC conjugate as done earlier [43]. Neutralizing antibody titers were determined by standard PRNT assay using LLCMK2 cells as before [42,43]. The antiserum dilution resulting in 50% reduction in plaque count (with reference to the number of plaques generated by the virus in the absence of antiserum), was expressed as the PRNT50 titer.

T cell assays

Spleens were harvested from mice ~7 weeks after the last immunization and cultured in 96-well plates for monitoring proliferative response and cytokine secretion in response to in vitro stimulation with DEN-2 and DEN-4 viruses (each at 0.03 PFU/cell). Cells were pulsed with [3H]-thymidine overnight at the end of 96 hours, and proliferation was quantitated by measuring the uptake of the radioisotope in a scintillation counter. IFN-γ and IL-4 were determined in splenocyte culture supernatants, at the indicated time points, using murine Quantikine cytokine ELISA kits (R & D systems), as described previously [42,45]. All assays for T cell responses were run in triplicate and values expressed as mean ± standard deviation (SD).

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