Clin Exp Vaccine Res.  2012 Jul;1(1):77-82. 10.7774/cevr.2012.1.1.77.

Recombinant influenza viruses as delivery vectors for hepatis B virus epitopes

• Song JM
• Lee KH
• Seong BL

• KMID: 2278791
• DOI: http://doi.org/10.7774/cevr.2012.1.1.77

Abstract

PURPOSE
Neuraminidase (NA) of influenza virus contains stalk region that shows a great deal of variability in both amino acid sequence and length. In this paper, we investigated generation of recombinant influenza viruses that had hepatitis B virus (HBV) B cell epitopes in the NA stalk region as a dual vaccine candidate.
MATERIALS AND METHODS
We used the WSH-HK reassortant helper virus for rescue of recombinant influenza virus containing HBV epitopes and reverse genetic protocol based on the use of micrococcal nuclease-treated virus cores for reconstitution of ribonucleoproteins.
RESULTS
We successfully generated a chimeric influenza viruses which contained 22 amino acid peptides in the stalk region derived from the surface and pre-surface protein HBV. The growth kinetics of the recombinant viruses was investigated after infection of Madin-Darby canine kidney (MDCK) and Madin-Darby bovine kidney (MDBK) cells and the rIV-BVPreS virus showed higher titer than other viruses in MDCK cells. We also confirmed the presence of HBV epitopes in the chimeric viruses by enzyme-linked immunosorbent assay (ELISA) using anti-HBV polyclonal antibody. When the ratio of recombinant virus verse wild type virus was calculated by ELISA, recombinant viruses exhibited 2 fold higher values than the wild type virus.
CONCLUSION
These results suggest that chimeric influenza virus which contained foreign antigens can be used as dual vaccine against both HBV and influenza viruses.

Keyword

NA stalk; Chimeric virus; Dual vaccine

MeSH Terms

Amino Acid Sequence
Enzyme-Linked Immunosorbent Assay
Epitopes
Epitopes, B-Lymphocyte
Helper Viruses
Hepatitis B virus
Herpesvirus 1, Cercopithecine
Influenza, Human
Kidney
Kinetics
Madin Darby Canine Kidney Cells
Neuraminidase
Orthomyxoviridae
Peptides
Viruses
Epitopes
Epitopes, B-Lymphocyte
Neuraminidase
Peptides

Figure

• 

  Fig. 1 Generation of recombinant influenza A virus. Reconstituted ribonucleoprotein (RNP) complex which has RNA in vitro transcribed with foreign epitopes and functional viral core proteins was transfected into Madin-Darby bovine kidney (MDBK) cells that had been infected with WSN-HK helper virus. Recombinant influenza A virus was selected by plaque assay on MDBK cells in the absence of protease and further amplified in MDBK cells. NA, neuraminidase; NP, nucleoprotein.

• 

  Fig. 2 Amino acid sequences of the neuraminidase (NA) stalk mutants from residues 36 to 80 of WSN NA. The B cell epitope sequences derived from HBV surface (rIV-BVS) and PreS2 (rIV-BVPreS2) protein are italicized. NAmut is the partial amino acid sequences of mutant NA on the intermediate cloning vector, pT3WSN-NAmut, for introducing foreign epitopes.

• 

  Fig. 3 Growth properties of recombinant influenza A viruses. rIV-BVS and rIV-BVPreS2 were cultured in Madin-Darby bovine kidney (MDBK) (A) and Madin-Darby canine kidney (MDCK) (B) cells for 72 hours then the virus titer of the sample was determined by plaque assay. PFU, plaque-forming unit.

• 

  Fig. 4 Binding properties between recombinant influenza A viruses and hepatitis B virus (HBV) antibody. rIV-BVS and rIV-BVPreS2 were coated on the microtiter plates and tested the binding activity of the anti-HBV polyclonal antibody by enzyme-linked immunosorbent assay.

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