Large-scale analysis of antigenic diversity of T-cell epitopes in dengue virus

BACKGROUND: Antigenic diversity in dengue virus strains has been studied, but large-scale and detailed systematic analyses have not been reported. In this study, we report a bioinformatics method for analyzing viral antigenic diversity in the context of T-cell mediated immune responses. We applied this method to study the relationship between short-peptide antigenic diversity and protein sequence diversity of dengue virus. We also studied the effects of sequence determinants on viral antigenic diversity. Short peptides, principally 9-mers were studied because they represent the predominant length of binding cores of T-cell epitopes, which are important for formulation of vaccines. RESULTS: Our analysis showed that the number of unique protein sequences required to represent complete antigenic diversity of short peptides in dengue virus is significantly smaller than that required to represent complete protein sequence diversity. Short-peptide antigenic diversity shows an asymptotic relationship to the number of unique protein sequences, indicating that for large sequence sets (~200) the addition of new protein sequences has marginal effect to increasing antigenic diversity. A near-linear relationship was observed between the extent of antigenic diversity and the length of protein sequences, suggesting that, for the practical purpose of vaccine development, antigenic diversity of short peptides from dengue virus can be represented by short regions of sequences (~<100 aa) within viral antigens that are specific targets of immune responses (such as T-cell epitopes specific to particular human leukocyte antigen alleles). CONCLUSION: This study provides evidence that there are limited numbers of antigenic combinations in protein sequence variants of a viral species and that short regions of the viral protein are sufficient to capture antigenic diversity of T-cell epitopes. The approach described herein has direct application to the analysis of other viruses, in particular those that show high diversity and/or rapid evolution, such as influenza A virus and human immunodeficiency virus (HIV)

Conservation and Variability of Dengue Virus Proteins: Implications for Vaccine Design

Dengue viruses (DENVs) circulate in nature as a population of 4 distinct types, each with multiple genotypes and variants, and represent an increasing global public health issue with no prophylactic and therapeutic formulations currently available. Viral genomes contain sites that are evolutionarily stable and therefore highly conserved, presumably because changes in these sites have deleterious effects on viral fitness and survival. The identification and characterization of the historical dynamics of these sites in DENV have relevance to several applications such as diagnosis and drug and vaccine development. In this study, we have identified sequence fragments that were conserved across the majority of available DENV sequences, analyzed their historical dynamics, and evaluated their relevance as candidate vaccine targets, using various bioinformatics-based methods and immune assay in human leukocyte antigen (HLA) transgenic mice. This approach provides a framework for large-scale and systematic analysis of other human pathogens

In silico Vaccine Design against Dengue Virus Type 2 Envelope Glycoprotein

Dengue fever is caused by the mosquito-borne virus termed (DENV). However, DENV-2 has been identified as the most prevalent amongst the Indonesian pediatric urban population, in contrast with the other four serotypes. Therefore, it is important to reduce severe infection risk by adopting preventive measures, including through vaccine development. The aim of this study, therefore is to use various in silico tools in the design of epitope-based peptide vaccines (T-cell and B-cell types), based on the DENV-2 envelope glycoprotein sequences available. Therefore, in silico methods were adopted in the analysis of the retrieved protein sequences. This technique was required to determine the most immunogenic protein, and is achieved through conservancy analysis, epitope identification, molecular simulation, and allergenicity assessment. Furthermore, B4XPM1, and KAWLVHRQW were identified from positions 204-212, while the 77 to 85 peptide region was considered the most potent T-cell and B-cell epitopes. The interaction between KAWLVHRQW and HLA-C*12:03 occurs with maximum population coverage, alongside high conservancy (96.98%) and binding affinity. These results indicated a potential for the designed epitopes to demonstrate high immunity against DENV-2

Characterization of a major neutralizing epitope on the yellow fever virus envelope protein using human recombinant monoclonal antibody fragments generated by phage display

Yellow fever virus (YFV) is a mosquito-transmitted, enveloped, positive stranded RNA virus belonging to the genus flavivirus, which causes hemorrhagic fever in humans in Africa and South America. The YFV is responsible for 200 000 clinical infections per year including 40 000 deaths. Despite the presence of a highly effective YF vaccine called 17D vaccine, this disease is now strongly re-emerging and has to be considered as a public health problem. The present live attenuated 17D vaccine has two major drawbacks: 1) the ancient production method by inoculating viable embryonated eggs which limits the vaccine production capacity and, therefore, impairs attempts to control the disease and may contribute to vaccine supply shortage. 2) this vaccine is a non clonal vaccine which is constituted of heterogenous virion sub-populations. Furthermore, recent reports of several cases of viscerotropic and neurotropic disease associated with 17D vaccination have raised the obvious question of vaccine safety. Taken together, these data show that it appears essential to design a new clonal vaccine which could be based on infectious cDNA clone and produced in animal cell culture. For this purpose, the knowledge of YFV neutralizing epitopes is essential. Because YFV immunity is mainly antibody-mediated, we wanted to isolate human neutralizing antibodies specific for YFV and use them as a tool to characterize the neutralizing epitopes of YFV. The phage display technology provides one of the most convenient systems to isolate such neutralizing recombinant antibody fragments. We generated YF patient-derived antibody phage libraries which were screened against purified virions of the YFV-204-WHO vaccine strain. This step led to the isolation of several single-chain antibody fragments (scFv) which recognized conformational and pH sensitive epitopes in the envelope E protein. Three genetically closely-related and competing scFvs were found to be able to neutralize in vitro the 17D vaccine strain and five wild-type African strains of YFV. To map their epitopes, neutralization escape variants of the YFV-17D-204-WHO were generated using one high-affinity scFv (scFv-7A). Amino acids (aa) E-153, E-154 and E-155 in domain I and aa E-71 in domain II of the E protein were shown to be the critical components of one complex neutralizing epitope. These aa do not form a contiguous epitope on the monomeric E protein, but are in close vicinity in the dimeric form the E protein is predicted to adopt, based on the crystal structures of related flaviviruses. The neutralizing epitope is thus predicted to be formed by contribution of aa from domain I and II of opposing E monomers. The nature of this epitope was supported by the analysis of one wild-type YFV strain (Senegal 90) which is naturally resistant to neutralization by scFv-7A. Microneutralization assays using sera from YFV-infected patients and 17D-immunized travelers confirm the importance of E-71 in YFV neutralization but also showed that those escape variants, originally present in the vaccine lot, do not carry a risk of neutralization escape in persons who are immunized with the 17D vaccine. The potential neutralization mechanism by which these scFvs act, particularly by preventing the fusion process, and their potential use as a therapeutical tool are discussed. The structural complexity of the epitope identified in this work has implications for understanding the mechanism of antibody-mediated neutralization of YFV and these data may be useful for the design of a new recombinant yellow fever vaccine based on a cDNA-derived infectious clone