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. 2016 Feb;24(2):375-384.
doi: 10.1038/mt.2015.210. Epub 2015 Nov 19.

Increased Valency of Conserved-mosaic Vaccines Enhances the Breadth and Depth of Epitope Recognition

Sultan Abdul-Jawad  1 , Beatrice Ondondo  1 , Andy van Hateren  2 , Andrew Gardner  1 , Tim Elliott  2 , Bette Korber  3 , Tomáš Hanke  4
Affiliations

Increased Valency of Conserved-mosaic Vaccines Enhances the Breadth and Depth of Epitope Recognition

Sultan Abdul-Jawad et al. Mol Ther. 2016 Feb.

Abstract

The biggest roadblock in development of effective vaccines against human immunodeficiency virus type 1 (HIV-1) is the virus genetic diversity. For T-cell vaccine, this can be tackled by focusing the vaccine-elicited T-cells on the highly functionally conserved regions of HIV-1 proteins, mutations in which typically cause a replicative fitness loss, and by computing multivalent mosaic proteins, which maximize the coverage of potential 9-mer T-cell epitopes of the input viral sequences. Our first conserved region vaccines HIVconsv employed clade alternating consensus sequences and showed promise in the initial clinical trials in terms of magnitude and breadth of elicited CD8(+) T-cells. Here, monitoring T-cells restricted by HLA-A*02:01 in transgenic mice, we assessed whether or not the tHIVconsv design (HIVconsv with a tissue plasminogen activator leader sequence) benefits from combining with a complementing conserved mosaic immunogen tHIVcmo, and compared the bivalent immunization to that with trivalent conserved mosaic vaccines. A hierarchy of tHIVconsv ≤ tHIVconsv+tHIVcmo < tCmo1+tCmo2+tCmo3 vaccinations for induction of CD8(+) T-cell responses was observed in terms of recognition of tested peptide variants. Thus, our HLA-A*02:01-restricted epitope data concur with previously published mouse and macaque observations and suggest that even conserved region vaccines benefit from oligovalent mosaic design.

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Figures

Figure 1
Figure 1
The vaccine immunogens and their expression in vitro. (a) Schematic representation of the vaccine immunogens showing 14 highly conserved regions of HIV-1 Gag (cyan), Pol (red), Vif (yellow), and Env (violet) proteins assembled into a chimeric protein with a human tissue plasminogen activator leader sequence (“t”) at the N-terminus (black) and Pk (blue), His (green) and C-myc (purple) tags recognized by mAbs at C-termini of proteins. tHIVconsv (conserved consensus) has HIV-1 sequences of the first-generation conserved region T-cell vaccines described by Létourneau et al. tHIVcmo (conserved mosaic) was designed as a mosaic complementing tHIVconsv for maximum coverage of group M HIV-1 isolates. Immunogens tCmo1, tCmo2, and tCmo3 were designed in silico as a trivalent mosaic over the same 14 regions as tHIVconsv and tHIVcmo. (b) All five genes coding for the above immunogens were inserted into a plasmid DNA, nonreplicating simian (chimpanzee) adenovirus ChAdV-63 and nonreplicating poxvirus MVA, and their expression was confirmed by immunofluorescence using fluoroscein isothiocyanate (FITC)-conjugated mAs (green) recognizing the C-terminal tags following transfection of DNA, or rChAdV63 and rMVA infection at multiplicity of infection 10 and 5, respectively, of HeLa cells. rMVAs expressing tHIVconsv and tHIVcmo were markerless for potential human use and the immunogens were detected by FITC-conjugated mAbs, MVA.tCmo1, MVA.tCmo2, and MVA.tCmo3 coexpressed GFP as a rescue marker, and the immunogens were detected by Alexa fluora 594-conjugated mAbs (red). Nuclei are stained using 4‘,6-diamidino-2-phenylindole (DAPI) (blue).
Figure 2
Figure 2
Coverage of global HIV-1 potential 9-mer T-cell epitopes (PTEs) present in the HIV Sequence Database. The graphs give the coverage of potential 9-mer PTEs of group M HIV-1s. Coverage is defined as the average number of potential epitopes per natural strain that are matched in the full M group sequence database per natural strain, within the bounds of the protein regions considered. Full-length proteins and the tHIVconsv region amino acid positions as in HXB2 are listed above the graphs. For the full-length proteins (Gag, Pol, Vif, and Env), the coverage by consensus and trivalent mosaic are shown in blue. For the conserved vaccine immunogens, the PTE coverages by the consensus as in the tHIVconsv, tHIVconsv+tHIVcmo and trivalent mosaic tCmo1+tCmo2+tCmo3 are shown in red/orange/yellow.
Figure 3
Figure 3
The breadth and depth of HLA-A*02:01 T-cell responses elicited by tHIVconsv and tHIVcmo vaccines alone or in a bivalent combination. Groups of 6-week-old female HHD mice were vaccinated intramuscularly with the tHIVconsv (black) or tHIVcmo (gray) vaccines alone, or combined at ½ (bottom-to-top stripes) or full (top-to-bottom stripes) doses each. Full dose were 100 µg of DNA, 108 infectious doses of rChAdV63 and 106 plaque-forming units of rMVA. (a) For all groups, a rChAdV63 prime-rMVA boost (CM) regimen was used, while (b) employed a DNA-DNA-DNA-rChAdV63-rMVA (DDDCM) regimen (Supplementary Table S1). Individual mouse splenocytes were tested for recognition of HLA-A*02:01-restricted epitope variants indicated below the graphs in an IFN-γ ELISPOT assay. Note that other A*02:01-restricted responses were not induced due to the immunodominance of the ones showed and the negative data are not shown. Results are presented as medians with interquartile range (n = 5) after subtracting for each animal the mean no-peptide background (dotted line). Immunogens matching precisely the tested epitopes are indicated on the x-axis below the peptides sequences.
Figure 4
Figure 4
Functionality of HLA-A*02:01 T-cell responses induced by mono-, bi-, and trivalent mosaic immunogens. Groups of 6-week-old female HHD mice were vaccinated iintramuscularly with either tHIVconsv vaccines alone (black), ½ doses of tHIVconsv+tHIVcmo (light grey) or ⅓ doses of tCmo1+tCmo2+tCmo3 (dark gray) vaccines using a CM regimen (Supplementary Table S1). The depth and breadth of elicited HLA-A*02:01-restricted T-cell responses were assessed for individual mice in IFN-γ ELISPOT assay (n = 6) (a), intracellular cytokine staining assay (n = 5) detecting IFN-γ, TNF-α, and CD107a (see Supplementary Figure S3 for representative gating strategy) (b) and killing assay (n = 8) (see Supplementary Figure S4 for gating strategy) (c). Results for responding peptides are shown as medians with interquartile range after subtracting for each animal the mean no-peptide background. Non-responding peptides are not shown. Immunogens matching precisely the tested epitopes are indicated on x-axis below the peptide sequences. Analysis of variance test was employed to determine statistically significant differences: * for P < 0.05; ** for P < 0.01; *** for P < 0.001.
Figure 5
Figure 5
Binding affinity of peptide variants to refolded recombinant HLA-A*02:01. For the two immunodominant epitopes, the relative peptide binding affinities to purified recombinant HLA-A*02:01 molecules were determined by a fluorescence polarization completion assay. Binding of TAMRA-labeled peptide FLPSDC*FPSV is reported in millipolarization units (mP) and is plotted as a percentage of the maximum polarization signal that was observed in the absence of competing unlabeled peptide. One of two experiments is shown.
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