The analysis of conservation of DENV antigens should consider both the pan-DENV antigenic diversity and the diversity between DENV serotypes. Furthermore, functional properties, such as HLA binding potential are essential for the assessment of immunogenic potential of antigens. DENV conservation analysis in previous studies was based on the traditional approach, in which a peptide is classified as conserved or not conserved based on analysis of each individual amino acid along with an arbitrary frequency threshold (typically 90% or higher). The premise of traditional conservation analysis of vaccine targets is that conserved epitope candidates are more likely to confer cross-protection between pathogen variants. We argue that variant inclusion is important for polyvalent coverage, since the array of factors making a peptide immunogenic is far too complex to assume that conserved predicted binders are automatically the best immunogens. Our systematic approach, that deploys analysis of conservation of blocks of peptides, has produced a 10-fold larger number of potential DENV T-cell epitope targets than the traditional approach. Similar to a previous benchmark study (Khan et al., 2008
), our method also enables vaccine target discovery that considers both the conservation of antigens and the immunogenic potential of these peptides. The peptide blocks determined in our study can be used to inform and focus the design of experimental studies of polyvalent dengue vaccines. Furthermore, our approach is applicable to any variable virus such as HIV, influenza, or Hepatitis C. It is also applicable to broader vaccine approaches such as identification of shared peptide targets across major Flavivirus
The block entropy analysis is an informed strategy for achieving broad strain coverage in vaccine design with the inclusion of significant but less frequent variants. Central to this approach is the fact that T-cell epitopes are recognized as peptides rather than single residues, and should therefore be analyzed as such. For example, the analysis of 9-mer blocks enables characterization of a set of peptides which collectively can be considered as conserved for immunological applications. In this study, we based the assessment of immunological potential using predictions of peptide binding to seven common HLA class I molecules for which prediction algorithms have been validated (Lin et al., 2008
). Some of the conserved blocks contain peptides which are all predicted to bind the same HLA allele – these blocks are considered to be immuno-functionally conserved. The peptide block thus becomes a unit of analysis for building combinatorial vaccine formulations with broad coverage of both pathogen variants and diverse HLA haplotypes. This analysis provides a reasonably sized set of targets that can be experimentally validated, for example by mass spectrometry (Reinhold et al., 2010
The premise for the concept of immuno-functional conservation is not only that all peptides in a block bind to MHC with the same affinity and HLA restriction, but also that there is enough redundancy in CTLs so that each epitope/MHC complex may elicit an equally strong T-cell response. However, immunodominance of certain epitopes and some T-cell receptors (TCRs; Nikolich-Zugich et al., 2004
) can lead to an uneven response to antigens upon vaccination and thus incomplete strain coverage upon challenge. High intra-block homology could allow for a population of CTLs to recognize all epitope in a block, which may also favor the concept of including entire immuno-functionally conserved blocks in a polyvalent vaccine construct. Hence, predicting the cross-protective capacity of a peptide-based vaccine gets more difficult as the number of T-cell epitope candidates increases. Furthermore, the larger the combinatorial space needed to cover the diversity of the four serotypes; the more complex the task to combine all of the epitopes in one vaccine without compromising its efficacy. Considering these factors, we choose to include all blocks in which five peptides or less cover 99% of the block. This number maybe subject to adjustment after proper experimental validation of the epitope pools.
Applying block entropy analysis to the proteomes of DENV1–4, yielded 1,732, 1,551, 1,394, and 1,245 conserved blocks of 8-, 9-, 10-, and 11-mer peptides respectively, as opposed to the results of the benchmark study (Khan et al., 2008
) which yielded 206, 165, 118, and 88 conserved 8-, 9-, 10-, and 11-mer peptides respectively, using the traditional criteria for conservation. Of the 1,551 blocks of 9-mer peptides, 110 blocks, consisting of 333 peptides, were predicted to be immuno-functionally conserved, based on their predicted binding affinity to HLA class I, which can form the basis of a T-cell-based polyvalent vaccine against DENV. The method presented here can be readily applied to other relevant viral pathogens such as influenza, HIV, or HPV, as well as extended to encompass MHC class II epitope candidates and functional B-cell epitopes.