Cell-Mediated Immune Responses to Smallpox Vaccination

Abstract

We report that vaccine dilution (1:1 or 1:10) and previous vaccinia virus vaccination status had no significant effect on cell-mediated immune responses (i.e., the immediate vaccinia virus-specific gamma interferonproducing T-cell response measured by enzyme-linked immunospot assay) 1 month after smallpox vaccination (Lancy-Vaxina; Berna Biotech, Switzerland).The evaluation of successful vaccination with vaccinia virus is based on visual confirmation of a dermal response (10). However, assessment of the effectiveness of the attenuated forms of smallpox vaccines (i.e., modified vaccinia Ankara strain, New York City Board of Health strain [NYCBH], or LC16m8) and the next generation of smallpox vaccines requires a broader understanding of the immune response to vaccinia virus (4). However, there are limited and sometimes conflicting data on the dependence of human cell-mediated immune responses to smallpox vaccination on vaccine dilutions (3, 5, 9) and previous vaccinia vaccination status (4,5,6). We have therefore evaluated whether vaccine dilutions or previous vaccination status affect the induction of cell-mediated immune responses to smallpox vaccination. Cell-mediated immune responses to smallpox vaccination were assessed as the immediate vaccinia virus-specific gamma interferon (IFN-␥)-producing T-cell response by enzyme-linked immunospot (ELISPOT) assays. The assay was performed as described previously (1, 7). Briefly, approximately 60 ml of venous blood was obtained from each volunteer in this trial just before vaccination (day 0) and 30 days after vaccination. Within 6 h of collection, peripheral blood mononuclear cells (PBMC) were isolated by using Ficoll-Hypaque density gradients. The PBMC were then resuspended at a concentration of 10 7 cells/ml in RPMI 1640-20% fetal bovine serum-10% dimethyl sulfoxide and were cryopreserved. The cryopreserved PBMC were thawed and washed once with RPMI 1640 supplemented with 10% fetal bovine serum and 50 U of Benzonase (Sigma-Aldrich)/ml. Cells were again washed and then resuspended with RPMI 1640 supplemented with 10% fetal bovine serum at a concentration of 5 ϫ 10 6 cells/ml. The prepared PBMC were infected for 1 h with the live vaccinia virus (fresh vaccinia virus obtained from lyophilized LancyVaxina vaccine) at a multiplicity of infection of 1. Cells were washed and added to 96-well ELISPOT plates (BD Biosciences Pharmingen) coated with anti-human IFN-␥ antibody (BD ELISPOT human IFN-␥ kit). Negative controls were uninfected cells with medium alone. Positive controls were uninfected PBMC stimulated with purified phytohemagglutinin (Sigma-Aldrich). Cells were cultured in duplicate wells at 5 ϫ 10 5 cells/well at 37°C for 18 h. Spots were counted by use of an automated microscope (Carl Zeiss MicroImaging, Inc., Germany) after the background value, obtained by using unstimulated cells, was subtracted. A postvaccination response for cell-mediated immunity was defined as positive if the vaccinia virus-specific IFN-␥-producing T-cell response by ELISPOT assays increased...

Real time PCR for the assessment of CD8+ T cellular immune response after prophylactic vaccinia vaccination

“…1B) therefore reveal a virus specific T cell effector repertoire prevaccination and a corresponding functional state 1 month after immunization that is comparable to data obtained from tetramer-and ELISPOT analysis (Ennis et al, 2002;Tenorio et al, 2004;Rock et al, 2006). It is interesting to note that cellular reactivity when assessed by molecular techniques 4 weeks after vaccination did not seem to be influenced with respect to concentrations of the smallpox vaccine or the status of previous vaccination (Rock et al, 2006;Kim et al, 2006;Kennedy et al, 2004). In addition similar results for CD8+ T cell response were monitored regardless of whether participants were vaccinia naïve or boosted after previous vaccination (Ennis et al, 2002;Dong and Denny, 2006).…”