Recombinant Vaccinia Virus-Induced T-Cell Immunity: Quantitation of the Response to the Virus Vector and the Foreign Epitope

Abstract

Recombinant vaccinia viruses (rVV) have been extensively used as vaccines, but there is little information about the total magnitude of the VV-specific T-cell response and how this compares to the immune response to the foreign gene(s) expressed by the rVV. To address this issue, we quantitated the T-cell responses to both the viral vector and the insert following the infection of mice with VV expressing a cytotoxic T lymphocyte (CTL) epitope (NP118-126) from lymphocytic choriomeningitis virus (LCMV). The LCMV epitope-specific response was quantitated by intracellular cytokine staining after stimulation with the specific peptide. To analyze the total VV-specific response, we developed a simple intracellular cytokine staining assay using VV-infected major histocompatibility complex class I and II matched cells as stimulators. Using this approach, we made the following determinations. (i) VV-NP118 induced potent and long-lasting CD8 and CD4 T-cell responses to the vector; at the peak of the response (ϳ1 week), there were ϳ10 7 VV-specific CD8 T cells (25% of the CD8 T cells) and ϳ106 VV-specific CD4 T cells (ϳ5% of the CD4 T cells) in the spleen. These numbers decreased to ϳ5 ؋ 10 5 CD8 T cells (ϳ5% frequency) and ϳ10 5 CD4 T cells (ϳ0.5% frequency), respectively, by day 30 and were then stably maintained at these levels for >300 days. The size of this VV-specific T-cell response was comparable to that of the T-cell response induced following an acute LCMV infection. (ii) VV-specific CD8 and CD4 T cells were capable of producing gamma interferon (IFN-␥), tumor necrosis factor alpha (TNF-␣), and interleukin-2; all cells were able to make IFN-␥, a subset produced both IFN-␥ and TNF-␣, and another subset produced all three cytokines. (iii) The CD8 T-cell response to the foreign gene (LCMV NP118-126 epitope) was coordinately regulated with the response to the vector during all three phases (expansion, contraction, and memory) of the T-cell response. The total number of CD8 T cells responding to NP118-126 were ϳ20-to 30-fold lower than the number responding to the VV vector (ϳ1% at the peak and 0.2% in memory). This study provides a better understanding of T-cell immunity induced by VV-based vaccines, and in addition, the technique described in the study can be readily extended to other viral vectors to determine the ratio of the T-cell response to the insert versus the vector. This information will be useful in optimizing prime-boost regimens for vaccination.Recombinant vaccinia viruses expressing foreign genes from different pathogens have been extensively used in various experimental studies and also in clinical trials (2,20). Despite the widespread use of vaccinia virus (VV) as a viral vector, there is little to no information available about the total size of the VV-specific T-cell response. The major impediment to such studies has been the absence of well-defined CD4 and CD8 T-cell epitopes of VV. This has prevented a detailed quantitative analysis of T-cell immunity to VV. Although there is information about the...

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