Figure 1.
Schematic representation of a 252-parameter FTA assay.
Splenocytes from mice were labeled with combinations of CTV (0 nM, 350 nM, 1295 nM, 4792 nM, 17729 nM and 65595 nM), CFSE (0 nM, 79 nM, 315 nM, 1106 nM 3859nm, 13505nm and 47269 nM) and CPD (0 nM, 106 nM, 690 nM, 2738 nM, 10262 nM and 38506 nM) to generate 252 discernable cell clusters. Cell clusters were pulsed with MHC-binding peptides (as outlined in Figure 3) to generate a panel of 42 peptide pulsed clusters and this repeated 6 times to generate 6 intra-animal repeats (i.e., 252 target cell clusters in total). Target cells were also labeled with DiI for discrimination from host splenocytes (not shown). (a) FTA cells were injected i.v. into host mice that had 6 days earlier been vaccinated with VV-HIV or left unvaccinated as a control. 18 hr after FTA injection, splenocytes were collected and target cells delineated from host splenocytes by DiI label using flow cytometry (not shown). (b) 2D plots of the fluorescence intensities of a panel of one replicate of the 42 peptide-pulsed clusters from the unvaccinated and vaccinated animals and an associated histogram analysis of clusters pulsed with titrations of the F2L CTL epitope, revealing killing in vaccinated animals. (c) An example of histogram analysis of the FTA T helper assay, with B220+ FTA cells pulsed with the Gag Th TH cell epitope being assessed for CD69 up-regulation from the vaccinated animal compared to those from the unvaccinated animal.
Figure 2.
The FTA assay can measure magnitude, functional avidity and epitope variant cross-reactivity of CTL responses in vivo.
Six BALB/c mice were vaccinated with 5×106 PFU VV Western Reserve i.p. A FTA was constructed using mouse splenocytes and comprised of fluorescent target cells pulsed with 6 different concentrations of the MHC-I binding peptides F2L, F2L mut, A52R, and HIV neg (as a negative control). FTA target cells were injected i.v. into infected mice 6 days post vaccination and after 18 hr in vivo % specific killing calculated for FTA target cells from harvested spleens. a) In vivo killing responses from six infected animals. b) Summary of responses from all mice with means of % specific killing and standard error of mean. c) Mean area under curve (AUC) measurements from % specific killing response curves and associated standard error of means. d) Mean effective concentration of peptides used to pulse target cells that generated half maximal responses (EC50) and associated standard error of means and P values.
Table 1.
HIV-1 pox virus vaccination regimes used in this study.
Figure 3.
High throughput screening of HIV-1 poxvirus vaccination regimes for high magnitude, high functional avidity and high epitope variant cross-reactive T cell responses in vivo.
Mice were vaccinated with 24 different vaccine regimes based on all combinations of FPV-HIV and VV-HIV administered either i.n. or i.m. in a prime-boost strategy as outlined in Table 1. Mice were vaccinated with 5×106 PFU of each vaccine and booster vaccinations were given 2 weeks after the priming vaccination. T cell responses were assessed using a 252-parameter FTA comprised of fluorescent target cells pulsed with 6 concentrations of the MHC-I binding peptides F2L, F2L mut, HIV Gag, HIV Gag mut, HIV Pol and HIV Env, and the MHC-II binding peptide Gag TH. FTA target cells were injected i.v. into vaccinated mice 6 days post vaccination and responses assessed after 18 hr in vivo using harvested spleens. % specific killing and TH cell activity was calculated as described in the Materials and Methods. a) Cumulative magnitude of T cell responses as AUC was plotted as a heat map depicting the range of highest (darkest colour) to lowest (lightest colour) TH cell (upper panel) or CTL (lower panels) responses. b) AUC heat maps of responses to the three HIV Gag epitopes. c) EC50 values of responses to the three HIV Gag epitopes. Values are only shown where EC50 values were calculable as described in the Materials and Methods. AUC and EC50 values are depicted as means from 6 intra-animal replicates. The results are representative of three independent experiments
Figure 4.
i.n.FPV-HIV/i.m.VV-HIV prime-boost vaccination improves the magnitude, functional avidity and epitope variant cross-reactivity of T-cell responses compared to prime or boost vaccinations alone.
Mice were vaccinated i.n. with FPV-HIV and/or i.m. VV-HIV. Mice were vaccinated with 5×106 PFU of each pox virus vaccine. Booster vaccinations were given 2 weeks post the previous vaccination. T-cell responses were assessed using 252-parameter FTAs as in Figure 3. a) % specific killing of FTA cells in vivo by CTL and b) TH cell activity induced by prime, boost, and prime-boost vaccination regimes, showing all 6 intra-animal replicate responses (upper panels) and means and standard error of means (lower panels) to the various CTL epitopes. b) Mean and standard error of means from a). AUC and EC50 values of: c) CTL responses and: d) TH cell responses. Values are only shown where EC50 values were calculable as described in the Methods. AUC and EC50 values are depicted as means from 5 intra-animal replicates. The results are representative of seven independent experiments.
Table 2.
Animal number requirements for screening assays.