A histochemical and DAPI staining of 293T cells transiently transfected with pTH.HIV_CONSV DNA (A), or infected with MVA.HIV_CONSV (B) or AdHu5.HIV_CONSV (C and D). HIV_CONSV protein expression was detected using mAb tag Pk at the C-terminus of the immunogen and a primary anti-Pk mAb followed by secondary FITC- (A and B) or AlexaFluor584- (C and D) conjugated detection antibodies. The AdHu5.HIV_CONSV vaccine also expressed GFP, which co-localized with the HIV_CONSV expression (D). (E) BALB/c mice were immunized using the regimen indicated below, and the HIV_CONSV-induced T cell responses were assessed in an ELISPOT assay using the H epitope. Results are shown as a mean±SD (n = 4). U–unimmunized; D–pTH.HIV_CONSV DNA; A–AdHu5.HIV_CONSV; and M–MVA.HIV_CONSV. For doses and timing, see Methods.

(A) Mice were immunized using the DAM regimen and the vaccine-induced responses were detected in an ex vivo ELISPOT assay. Results are shown as a mean±SD (n = 4). For doses and timing, see Methods. (B) Identified epitope peptides and their origin. (C) Killing of murine EL4 A2-K^d (top) and human JK A2-K^d (bottom) target cells sensitized with the shown peptides in a ⁵¹Cr-release assay after a 5-day in vitro peptide re-stimulation. Black, grey and white bars indicated effector to target ratios of 100, 50 and 25 to 1, respectively.

(A) Localization of the 14 most highly conserved regions of the HIV-1 proteome. The numbers written vertically under each fragment boundary indicate the first and last aa positions using the HXB2 reference strain numbering (http://www.hiv.lanl.gov/content/hiv-db/LOCATE/locate.html). (B) Predicted aa sequence of the the HIV_CONSV immunogen with indicated fragment numbers. (C) Summary of the fragments including: the fragment number; the protein in which it was embedded; the clade of the consensus sequence selected for inclusion in the immunogen, alternating between clades A–D; additional clades that have identical HIV_CONSV; and position numbers in the chimeric vaccine. The number of additional clades with identical consensus sequences to selected clade reflects the high level of conservation in these regions, and is encouraging in terms of the global potential of the vaccine. The consensus sequences compared were to the M group consensus, clades A–K, and three very common recombinant circulating forms CRF01 (common in Asia and Africa), CRF02 (common Africa), CRF08 (common in China) retrieved from the Los Alamos database 2004 consensus alignment (http://www.hiv.lanl.gov/content/hiv-db/CONSENSUS/M_GROUP/Consensus.html). (D) Schematic representation of the HIV_CONSV immunogen (not drawn to scale) indicating clade anternation (above), overlapping peptide pool derivation and protein origin by colour coding. (E) Hamming distances between the HIV_CONSV antigen fragments and the global circulating viral sequences. The full M group alignment, including recombinant sequences, was used for the comparison. The Los Alamos database alignment contains only one sequence person, and contains sequences from between 600 and 1000 individuals in these proteins. The Hamming distance range for 95% of the sequences relative to the vaccine immunogen is given by the vertical lines. The distances between the full length natural proteins were then calculated relative to HXB2 reference strain Env, Vif, Gag and Pol sequences for comparison. Distance measures are minimal estimates, as gaps inserted in regions where insertions and deletions occur were not counted. (F) Numbers of known CD8⁺ T cell epitopes (defined to within 12 aa or less in the Los Alamos HIV-1 database) in each of the 14 conserved protein fragments included in the HIV_CONSV immunogen are shown. When more than one HLA class I presenting molecules can present the same HIV-1 epitope, then each is counted as a distinct epitope; if more than one sequence variant has been described as an epitope presented by the same class I molecule, then these are counted as a distinct epitopes; however, if an HLA serotype and genotype that are potentially the same are each described as presenting the same epitope (like A2 and A*0201) they are scored as a single epitope.

Mice were immunized using the regimen and immunogen indicated above (A, B and C) or below (D) the graphs and the HIV_CONSV-specific responses were determined in ex vivo ELISPOT (A and E) or ICS (B and D) assays detecting the indicated cytokines and using for restimulation overlapping peptide pools schematically shown in Fig. 1D (A and B) or individual epitope peptides (D and E). (C) Identified peptides or epitope sequences and their origin, name and T cell reactivity. In (D): white–IFN-γ; black–IL-2; stripy-IFN-γ+IL-2; and grey–TNF-α; *-responses significantly above the no-peptide background (p<0.05). In (E): white–no peptide followed from left to right by epitopes H, G1, G2, P1, P2 and P3. Results are shown as a mean±SD (n = 4). For doses and timing, see Methods.

The HIV_CONSV-specific memory T cells were assessed in healthy and HIV-1-infected subjects using an IFN-γ ELISPOT assay after a 10-day peptide and cytokine culture. (A) Summed frequencies of HIV_CONSV-specific cells detected in healthy (n = 9) and HIV-1-infected (n = 13) subjects. The bars show the group medians of 578 SFU/10⁶ and 8,092 SFU/10⁶ cells for the healthy and infected subjects, respectively. (B) In five subjects indicated below, cultured PBMC were left undepleted (grey) or depleted of CD8⁺ cells (black) prior to the ELISPOT assay. The difference between undepleted (median = 8,092 SFU/10⁶ cells) and CD8-depleted samples (median = 550 SFU/10⁶ cells) was statistically significant (p = 0.0313). (C) Responses to individual HIV_CONSV-derived peptide pools as shown in Fig. 1D determined for the HIV-1-infected (grey) and healthy (black) subjects shown as medians. (D) Responses to individual peptides pools for each HIV-1-infected patient indicated below. Bars show a mean±SD of three assay wells and ‘*’ indicates a positive response according to criteria set in Methods. Due to sample shortage, subject 021 was not tested.