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Comparative Study
. 2006 Jul;80(13):6259-66.
doi: 10.1128/JVI.00084-06.

Immunogenicity of cytopathic and noncytopathic viral vectors

Gabriela Plesa  1 , Philip M McKenna, Matthias J Schnell, Laurence C Eisenlohr
Affiliations
Comparative Study

Immunogenicity of cytopathic and noncytopathic viral vectors

Gabriela Plesa et al. J Virol. 2006 Jul.

Abstract

The impact of cytolytic versus noncytolytic viral infections on host responses is not well understood, due to limitations of the systems that have been used to address this issue. Using paired cytopathic and noncytopathic rabies viruses that differ by only two amino acids, we investigated several fundamental aspects of the immune response to these viral vectors. Greater cytopathic capacity translated into a greater degree of cross-priming to CD8(+) T cells (T(CD8)(+)) and more-robust short-term humoral and cellular responses. However, long-term responses to the two viruses were similar, suggesting that direct priming drives the bulk of the T(CD8)(+) antirabies response and that enhanced acute responses associated with greater virally mediated cellular destruction were balanced by other factors, such as prolonged antigen expression associated with noncytopathic virus. Such compensatory mechanisms may be in place to ensure comparable immunologic memories to various pathogens.

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Figures

FIG. 1.
FIG. 1.
Construction of cytopathic and noncytopathic recombinant RV vectors expressing different model antigens. VSVwtM or mutated VSV M was introduced between the N and P genes of RVnoM, resulting in RVwtM (cytopathic phenotype) or RV*M (noncytopathic phenotype). The Ova257-264 epitope embedded within the influenza nucleoprotein (NP/S) was inserted between rabies G and L genes (RVnoM-NP/S, RVwtM-NP/S, or RV*M-NP/S).
FIG. 2.
FIG. 2.
Analysis of cytolytic capacities of recombinant RVs. (a) Live cells counted by trypan blue exclusion. MC57G cells were infected with recombinant RVs at an MOI of 1, and trypan-excluding cells were counted at different times postinfection. Data are representative of three independent experiments, each of them done in triplicate. (b) MTT assay. Three days after the MC57G cells were infected with recombinant RVs at an MOI of 1, cell viability was measured by MTT conversion as described in Material and Methods. Data are representative of four independent experiments done in duplicate. The numbers represent P values calculated using Student's t test. (c) Annexin and PI staining of MC57G cells infected with recombinant viruses at an MOI of 1 and analyzed at the indicated time points. The numbers represent percentages of cells corresponding to each quadrant. The experiment was done twice. OD, optical density; PI, propidium iodide.
FIG. 3.
FIG. 3.
One-step growth curves of recombinant RVs. MC57G cells were infected with recombinant viruses at an MOI of 10. Aliquots of culture supernatants were collected at different time points, and viral titers were determined in duplicate. Data are representative of four independent experiments.
FIG. 4.
FIG. 4.
Recombinant RVs express variable levels of Ova257-264 epitope. MC57G cells were infected with RVnoM-NP/S, RVwtM-NP/S, or RV*M-NP/S at an MOI of 1 in triplicate, and at different times postinfection, the cells were stained with an anti-Kb/Ova257-264 antibody followed by a secondary FITC-labeled antibody and analyzed by flow cytometry for the mean fluorescence intensity (MFI). Three separate experiments showed similar results. P values (shown above bars) were calculated using Student's t test.
FIG. 5.
FIG. 5.
Cross-priming of recombinant RVs. P815 cells were infected with the recombinant viruses at an MOI of 3 for 2 days and then UV irradiated. Cells were injected i.p. into C57BL/6 mice, and 7 days later, the Ova257-264-specific TCD8+ responses were assayed by ELISPOT assay for IFN-γ-positive cells. The data represent three experiments with two mice per group. For each experiment, the maximal response was set at 100%, and the other responses were related to the maximal value. The variances for the two groups were sufficiently similar (0.039 and 0.037) to allow application of a paired Student t test assuming equal variance.
FIG. 6.
FIG. 6.
In vivo rabies and Ova257-264-specific immune responses induced by recombinant RVs. C57BL/6 mice were infected i.p. with 104 focus-forming units of recombinant RVs as indicated. Mice were bled 8 (a) and 28 (b and c) days after infection, and antibody titers specific for RVG (a and b) and RVN (c) were determined by ELISA. (d) The frequency of rabies-specific, IFN-γ-producing TCD8+ cells was determined at day 8 and 28 postinfection by ELISPOT assay using MC57G cells infected with recombinant RV*M. (e) Ova257-264-specific TCD8+ responses were analyzed by ELISPOT assay at 28 days postinfection by using MC57G cells pulsed with Ova257-264 peptide. For each graph, the bars represent the means for three mice per group. Data are representative of three identical experiments. OD, optical density; Neg, negative.
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