A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice - PubMed (original) (raw)

A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice

M Singh et al. J Virol. 1999 Jun.

Abstract

It has been shown previously that measles virus (MV) can be successfully used to express foreign proteins (M. Singh and M. A. Billeter, J. Gen. Virol. 80:101-106, 1998). To develop an inexpensive MV-based vaccine, we generated recombinant MVs that produce structural proteins of hepatitis B virus (HBV). A recombinant virus that expressed the HBV small surface antigen (HBsAg) was analyzed in terms of its replication characteristics, its genetic stability in cell culture, and its immunogenic potential in genetically modified mice. Although this virus showed a progression of replication slightly slower than that of the parental MV, it appeared to stably maintain the added genetic information; it uniformly expressed the appropriately glycosylated HBsAg after 10 serial passages. Genetically modified mice inoculated with this recombinant MV produced humoral immune responses against both HBsAg and MV proteins.

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Figures

FIG. 1

FIG. 1

Cloning HBsAg and HBcAg ORFs in p(+)MVNSe antigenomic MV plasmid. ORFs of MV and HBV are shown as rectangles (not to scale) labeled with letters as follows: N, nucleocapsid; P, phosphoprotein; M, matrix; F, fusion; H, hemagglutinin; and L, large protein of MV. Stippled rectangles denote nontranslated regions, and vertical bars denote the nontranscribed intergenic trinucleotides. The triangle labeled “aigr” represents the artificial intergenic region, which consists of gene termination, intergenic, and gene start sequences followed by unique cloning sites. The flanking sequences together with start and stop codons (underlined) of the HBsAg ORF, plasmid names together with total sizes in base pairs, MV antigenomic nucleotide numbers (based on EMBL accession no. Z66517), and restriction sites are as indicated. T7 indicates the T7 RNA polymerase promoter, δ indicates the hepatitis delta virus ribozyme, and TΦ indicates the T7 RNA polymerase terminator.

FIG. 2

FIG. 2

Expression of HBsAg and HBcAg from recombinant MVs. Vero cells were infected with either MVHBs, MVHBsc, or MV-tag-Edm (indicated as MV) and processed for indirect immunofluorescence as described in Materials and Methods. HBVsAg-specific (a) and HBVcAg-specific (b) signals were detected with goat anti-HBsAg and rabbit anti-HBcAg antibodies, respectively, followed by anti-goat- and anti-rabbit antibody–FITC conjugate, respectively.

FIG. 3

FIG. 3

Western immunoblots of HBsAg and HBcAg expressed from recombinant MVs. Vero cells were infected with either MVHBs, MVHBsc, or MV-tag-Edm (indicated as MV), and total cellular proteins were harvested at 24 hpi and processed for Western blots as described in Materials and Methods. The nylon membranes with transferred proteins were developed with goat anti-HBsAg antibodies followed by anti-goat antibody–HRPO conjugate (a) and with rabbit anti-HBcAg antibodies followed by anti-rabbit antibody–HRPO conjugate (b), and proteins were visualized by enhanced chemiluminescence. Two protein bands of approximately 27 and 24 kDa observed in MVHBs cell lysates and a single band of approximately 21 kDa observed in MVHBsc cell lysates are marked with arrowheads. The molecular mass markers (in kilodaltons) are indicated.

FIG. 4

FIG. 4

Growth curve comparison of MVHBs and MV-tag-Edm. Vero cells were infected with MVHBs or MV-tag-Edm at an MOI of 0.01, and the virus was harvested at the indicated time points. The virus titers, expressed as TCID50s per milliliter, are taken from one of three independent experiments; the variations (which were minor) are not shown.

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References

    1. Albrecht P, Herrmann K, Burns G R. Role of virus strain in conventional and enhanced measles plaque neutralization test. J Virol Methods. 1981;3:251–260. - PubMed
    1. Cattaneo R, Rebmann G, Schmid A, Baczko K, ter Meulen V, Billeter M A. Altered transcription of a defective measles virus genome derived from a diseased human brain. EMBO J. 1987;6:681–688. - PMC - PubMed
    1. Cattaneo R, Will H, Darai G, Pfaff E, Schaller H. Detection of an element of the SV40 late promoter in vectors used for expression studies in COS cells. EMBO J. 1983;2:511–514. - PMC - PubMed
    1. Chen R T, Markowitz L E, Albrecht P, Stewart J A, Mofenson L M, Preblud S R, Orenstein W A. Measles antibody: reevaluation of protective titers. J Infect Dis. 1990;162:1036–1042. - PubMed
    1. Cornu T. Use of measles virus as a vector: stable expression of a supplementary large transcription unit or additional foreign viral glycoproteins. M.S. thesis. Zurich, Switzerland: University of Zurich; 1997.

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