Direct comparison of antigen production and induction of apoptosis by canarypox virus- and modified vaccinia virus ankara-human immunodeficiency virus vaccine vectors - PubMed (original) (raw)

Comparative Study

. 2007 Jul;81(13):7022-33.

doi: 10.1128/JVI.02654-06. Epub 2007 Apr 4.

Affiliations

Comparative Study

Direct comparison of antigen production and induction of apoptosis by canarypox virus- and modified vaccinia virus ankara-human immunodeficiency virus vaccine vectors

Xiugen Zhang et al. J Virol. 2007 Jul.

Abstract

Recombinant poxvirus vectors are undergoing intensive evaluation as vaccine candidates for a variety of infectious pathogens. Avipoxviruses, such as canarypox virus, are replication deficient in mammalian cells by virtue of a poorly understood species-specific restriction. Highly attenuated vaccinia virus strains such as modified vaccinia virus Ankara (MVA) are similarly unable to complete replication in most mammalian cells but have an abortive-late phenotype, in that the block to replication occurs post-virus-specific DNA replication. In this study, an identical expression cassette for human immunodeficiency virus gag, pro, and env coding sequences was placed in canarypox virus and MVA vector backbones in order to directly compare vector-borne expression and to analyze differences in vector-host cell interactions. Antigen production by recombinant MVA was shown to be greater than that from recombinant canarypox virus in the mammalian cell lines and in the primary human cells tested. This observation was primarily due to a longer duration of antigen production in recombinant MVA-infected cells. Apoptosis induction was found to be more profound with the empty canarypox virus vector than with MVA. Remarkably, however, the inclusion of a gag/pro/env expression cassette altered the kinetics of apoptosis induction in recombinant MVA-infected cells to levels equal to those found in canarypox virus-infected cells. Antigen production by MVA was noted to be greater in human dendritic cells and resulted in enhanced T-cell stimulation in an in vitro antigen presentation assay. These results reveal differences in poxvirus vector-host cell interactions that should be relevant to their use as immunization vehicles.

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Figures

FIG. 1.

FIG. 1.

HIV protein expression by vCP205 and MVA205. (A) Western blot of HIV antigen expression in infected CEFs. Cells were harvested 24 h following infection with test viruses. Cells were harvested (C) and supernatants pelleted (S) for detection of virus-like particle production. The blot was probed using HIV-positive patient sera. The positions of molecular mass markers are shown on the left. (B) HeLa cells were infected and treated as described for panel A.

FIG. 2.

FIG. 2.

Gag-Env pseudovirion production and release by recombinant poxviruses in mammalian adherent cell lines. Each cell line shown was infected at an MOI of 10 with vCP205 or MVA205. Cellular supernatants were harvested at multiple time points following infection, and p24 antigen was measured by an antigen capture ELISA. p24 antigen production by MVA205 is indicated by black squares, and antigen production by vCP205 is shown by open circles. Error bars represent standard deviations for triplicate wells.

FIG. 3.

FIG. 3.

Gag-Env pseudovirion production and release from suspension cells and primary cells. Suspension cells were infected at an MOI of 10 with vCP205 or MVA205, and supernatants were collected at the indicated times for p24 antigen measurement. Note that the _y_-axis scale is different from that in Fig. 2. p24 production by MVA205 is shown by black squares, and vCP205 production is indicated by open circles. Error bars represent standard deviations for triplicate wells.

FIG. 4.

FIG. 4.

Cellular versus supernatant antigen expression. Cells were infected at an MOI of 10 with either virus and harvested at the indicated times for p24 antigen quantitation for cells (left) and the corresponding pelleted supernatants (right). Black squares indicate MVA205, and white circles indicate vCP205. Error bars represent standard deviations for triplicate wells. (Top) HeLa cells. (Bottom) TE671 (human rhabdomyosarcoma) cells.

FIG. 5.

FIG. 5.

Comparison of efficiencies of infection by canarypox virus and MVA. Each of the cell types used in this study was infected with vCP-GFP and MVA-GFP, and the percentages of cells expressing GFP were quantified by flow cytometry. Gray histograms represent uninfected cells, and white histograms represent green fluorescence 24 h following infection. Bars and corresponding numbers indicate percentages of the infected cell populations expressing GFP. Suspension cells with lower infection efficiencies are shown in panels K and L. Results are representative of experiments performed three times.

FIG. 6.

FIG. 6.

Measurement of virus-mediated apoptosis. Three methods were used to assess apoptosis in virus-infected HeLa cells. (A) DNA fragmentation was assessed in cells infected at the indicated times. The DNA marker was lambda DNA digested with HindIII and EcoRI. The positive control was a kit positive control from the manufacturer (Roche), and the negative control sample was from uninfected cells. (B) ELISA for detection of histone-bound DNA in cytoplasm. MVA205, vCP205, and vCP1452 were evaluated in HeLa cells for apoptosis at 12 and 24 h. Blank, no added DNA; Neg Ctr, DNA from uninfected cells; Camp, positive control from camptothecin-treated HeLa cells. (C) HeLa cells were infected with the indicated viruses and harvested at 12 or 24 h for annexin V and PI staining by flow cytometry. Gates were set on single-stained uninfected cell populations. Numbers represent percentages of cell populations in each quadrant.

FIG. 7.

FIG. 7.

Effect of insert on virus-induced apoptosis. HeLa cells were infected with the indicated viruses and harvested at 12 or 24 h for annexin V and PI staining by flow cytometry. Gates were set on single-stained uninfected cell populations. (A) Negative control, (B) camptothecin, (C) MVA/T7, (D) MVA empty vector, (E) MVA205, (F) MVA-GFP, (G) canarypox virus empty vector, (H) vCP205, (I) vCP-GFP. Numbers represent percentages of the cell populations in each quadrant.

FIG. 8.

FIG. 8.

Quantitative real-time PCR for viral DNA. Quantitation of viral DNA was performed using primers within the HIV gag gene compared with a purified DNA standard. Time zero represents input viral DNA. Gray squares represent DNA copies in MVA205-infected cells, and white circles represent DNA copies in vCP205-infected cells.

FIG. 9.

FIG. 9.

Infection of human DC and in vitro antigen presentation. (A) Expression of HIV Env and Gag proteins in DC loaded with vCP205 (left) or MVA205 (right), as assessed by flow cytometry. The percentage of cells infected at an MOI of 5 is shown by detection of the respective vector. Data shown are the means for six experiments plus standard deviations. (B to D) IFN-γ ELISPOT analysis of T-cell responses following coculture with DC infected with vCP205, MVA205, or a vector controls (CPpp and MVAp580, respectively). Results from experiments with blood cells from three representative donors are shown. DC were infected at an MOI of 5 unless noted otherwise.

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