Dengue virus epitopes

National Center for Biotechnology Information , U. J Infect Dis. Published online Jan James S. Author information Article notes Copyright and License information Disclaimer. Corresponding author.

Received Jun 29; Accepted Sep All rights reserved. For Permissions, please e-mail: journals. This article has been cited by other articles in PMC. Abstract Dengue fever and dengue hemorrhagic fever are significant global public health problems, and understanding the overall immune response to infection will contribute to appropriate management of the disease and its potentially severe complications.

Peptide Validation by Synthetic Peptides Synthetic peptides for validating the peptides identified in this study were obtained from Genscript Corporation.

Open in a separate window. Figure 1. Table 1. Figure 2. Figure 3. Figure 4. Supplementary Data: Click here to view. Notes Acknowledgments. Financial support. Potential conflict of interest. All authors: No reported conflicts. References 1. Guzman A, Isturiz RE. Update on the global spread of dengue. Int J Antimicrob Agents. World Health Organization. Report of the Scientific Working Group on Dengue.

Geneva: WHO; Rothman AL. Dengue: defining protective versus pathologic immunity. J Clin Invest. Dengue vaccines: problems and prospects. Indian J Med Res. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol. Heinz FX. Epitope mapping of flavivirus glycoproteins. Adv Virus Res. Dengue vaccines regulatory pathways: a report on two meetings with regulators of developing countries.

PLoS Med. Viremia and immunogenicity in nonhuman primates of a tetravalent yellow fever-dengue chimeric vaccine: genetic reconstructions, dose adjustment, and antibody responses against wild-type dengue virus isolates. Am J Trop Med Hyg. Inoculation of plasmids expressing the dengue-2 envelope gene elicit neutralizing antibodies in mice.

Milich DR. Synthetic T and B cell recognition sites: implications for vaccine development. Adv Immunol. Barrett AD. Current status of flavivirus vaccines. Ann N Y Acad Sci. Clinical proof of principle for ChimeriVax: recombinant live, attenuated vaccines against flavivirus infections.

Dengue virus type 1 DNA vaccine induces protective immune responses in rhesus macaques. J Gen Virol. Synthetic peptides: a next generation of vaccine? Immunol Today. Arnon R, Horwitz RJ. Synthetic peptides as vaccines.

Curr Opin Immunol. Immune response to synthetic peptides of dengue prM protein. Mapping of biologically active helper T-cell epitopes on the flavivirus envelope glycoprotein.

Vaccines T-helper cell epitopes on the E-glycoprotein of dengue 2 Jamaica virus. Halstead SB. In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody. Dengue viruses and mononuclear phagocytes.

To probe the phenotype of virus-specific T cells, epitopes derived from viral sequences need to be known. DENV infection is associated with a range of clinical manifestations, from asymptomatic to more severe presentations including dengue hemorrhagic fever DHF and dengue shock syndrome DSS.

There is currently no specific therapy available for the treatment of dengue diseases other than supportive care. Sridhar et al. In order to develop effective DENV therapeutics and vaccines, it is important to define immunological correlates of protection against DENV infection as well as biomarkers that can be used to access their safety and efficacy. Although T cells have important functions in combating viral pathogens, both pathological and protective effects of T cells have been reported in the context of DENV infection 9 — According to T cell original antigenic sin, cross-reactive T cells that are specific for a primary DENV serotype become predominant during a secondary heterologous infection 9 — Consequently, the expansion of preexisting cross-reactive and low-affinity memory T cells results in ineffective viral control and contributes to immunopathology and severe dengue disease through excessive production of inflammatory cytokines 9 — In contrast to the implications of original antigenic sin, several lines of evidence indicate that T cells contribute to the control of DENV infection.

Furthermore, HLA alleles associated with protection from severe dengue disease are also associated with strong and multifunctional T cell responses, supporting the notion that T cells have protective functions during DENV infection 25 — The main characteristic of an efficient vaccine is the prophylactic effect provided by protective neutralizing antibodies.

Indeed, a single dose of the live attenuated tetravalent DENV vaccine TV provides complete protection against infection with a DENV-2 challenge virus 31 , potentially highlighting the importance of harnessing the protective functions of both humoral and cellular antiviral immunity.

Human antigen-specific T cell immune responses are driven by two factors that are host specific. First the capability of antigen-derived peptides to be bound and presented in the context of HLA class I and II molecules.

Second, the immunogenicity of those peptides that depends on the capability of T cells to recognize through T cell receptor TCR the HLA-peptide complex and trigger T-cell specific immune responses. In this review, we summarize previous published data of all the DENV-epitopes experimentally identified by us and others by performing an overall analysis of data available in Immune Epitope Database www.

This query retrieved a total of 57 different publications Table 1. Most of the studies were focused on NS3 protein or multiple DENV proteins defined as immunodominant region based on previous studies Table 1. Specifically, we considered human samples derived from different geographical locations and collected from either healthy DENV seropositive blood donors DENV 25 , 26 , 28 , 73 , 77 , 80 or patients during acute dengue infection 29 , 74 , 75 or healthy donor after experimental vaccination with dengue virus 76 , 78 , 81 Table 1.

A total of epitopes were described in the 57 references retrieved by the query Table 1 ; of those, were restricted by HLA class I molecules, and epitopes were restricted by HLA class II. Epitopes derived from those 14 studies have been extracted from IEDB, redundant epitopes independently defined in donors with different HLA restriction have been included to give prominence to promiscuous peptides able to be presented and recognized by different HLA molecules.

DENV T cell immunodominance. A HLA class I restriction. NS3 immunodominance is confirmed throughout all the different studies used for the analysis, representing the most frequent target of CD8 T cell response, disregarding geographical location, and HLA restriction.

Capsid immunodominance is consistently identified in all the different studies analyzed, representing the most frequent target of CD4 T cell response. Finally, protein immunodominance for both CD4 and CD8 T cells is also function of the multiple exposure of DENV infection, that tends to skew protein immunodominance toward epitopes highly conserved across the different DENV serotypes as previously reported Overall, T cell protein immunodominance is quite complex and widely focused on different protein targets, suggesting that in order to trigger an efficient DENV-specific T cell response both Structural C, prM, Envelope and Non-Structural NS proteins are required.

Epitope identification studies have provided the basis for phenotyping DENV-specific T cell responses directly ex vivo without the need for in vitro stimulation that could potentially alter T cell phenotypes. Utilizing the knowledge of the epitopes recognized we developed the megapool approach, which allows for combining a large number of peptides into one peptide pool based on sequential lyophilization.

These peptides are pooled, lyophilized, and resuspended to form a master mix, which is then used to stimulate T cell ex vivo More recent studies using DENV peptide pools shows that higher magnitude and more polyfunctional CD8 T cell responses correlate with HLA alleles that are associated with reduced risk of severe dengue disease 27 , 28 , which is consistent with the report that the frequency of DENV-specific cytokine-producing CD8 T cells is higher among children who subsequently developed subclinical secondary infection than those who developed symptomatic secondary infection Since DENV infection initiates at the site of the mosquito bite in the host skin, it is possible that CD8 T cells may migrate to the site of infection and mediate localized responses.

Moreover, DENV-specific CD8 T cells are readily detectable in the skin of DENV-infected individuals at the acute stage 67 , suggesting that these cells may exert effector functions at the site of infection. Tissue-resident memory T Trm cells reside in non-lymphoid tissues including the skin and can serve as a front line of defense against invading pathogens such as vaccinia virus It would be interesting to investigate whether DENV-specific CD8 T cells could differentiate into Trm cells in the skin that may mount rapid and localized protective immunity upon reinfection.

Chandele et al. DENV-specific Tem and especially Temra cells display specialized gene expression profiles and upregulated genes that are associated with activation, co-stimulation, and effector functions 90 , which is consistent with the previous study from Chandele et al.

In addition, DENV-specific CD4 T cells with cytotoxic activity have been reported by numerous studies 12 and their frequency may be lower in patients with more severe dengue disease Notably, CX3CR1 has recently been reported to be a member of a gene set that can predict severe dengue disease Subsequent transcriptomic profiling studies further revealed the gene expression patterns and heterogeneity of CD4 Temra cells and identified additional phenotypic markers such as GPR56 and CD that are specifically expressed by cytotoxic CD4 Temra cells and confirmed their expression on DENV-specific Temra cells 98 , Additionally, cytotoxic CD4 Temra cells may have undergone extensive clonal expansions based upon TCR analysis 98 , 99 , supporting the notion that these cells are induced by repeated DENV infections.

However, their functional significance in the context of DENV infection is less well-defined It has been reported that the frequency of Treg cells and the ratio of Treg cells to effector T cells are significantly higher during acute DENV infection than after recovery in patients with mild disease but not in those with severe disease However, subsequent studies indicate that the frequency of Treg cells is not associated with viral load or disease severity Therefore, whether and how Treg and Tr1 cells influence antiviral immune response and disease progression during DENV infection warrants further investigation.

Tfh cells have been associated with protective roles in human infectious disease — and vaccinees — They provide several forms of T cell help to B cells such as signals that promote survival, proliferation, plasma cell differentiation, hypermutation, class-switch recombination, adhesion and chemoattraction cell migration Tfh cells are essential for the generation of most isotype switched and affinity matured antibodies, and therefore they have an obvious role in protective immunity against pathogens.

A recent breakthrough has been the ability to detect Tfh cells in peripheral blood , thus allowing their assessment in PBMC samples based on surface markers. Recent studies have reported an expansion of peripheral Tfh cells in DENV-infected children during the acute phase Furthermore, Tfh cells are more abundant in patients with secondary DENV infection and in those who developed a more severe dengue disease Although Tfh cells has been shown to promote DENV-specific antibody responses in mice , the differentiation and functional significance of DENV-specific Tfh cells in humans warrants further investigation.

All DENV-specific T cell phenotypes discussed in this review as well as the markers they express are summarized in Figure 2 and Table 2 , respectively. Comprehensive epitope identification over the last few years has provided the tools that allow one to probe for DENV specific T cell responses in donors exposed to natural infection and vaccination. Global assessment of dengue virus-specific CD4 and CD8 T cell responses in dengue-endemic areas led to the development of new tools megapools that allow analysis of small samples typically available from pediatric and hospital cohorts.

It has also been demonstrated that DENV-specific CD4 and CD8 responses are more complex than previously thought, with different subsets revealed by in depth phenotypic and transcriptomic analyses.

The hypothesis that these different subsets have unique roles and dictate and shape clinical outcomes and vaccine efficacy will have to be explored in the near future. DENV specific T cell phenotypes. Tissue resident CD8 T cells patrol skin for infected antigen presenting cells APCs and can generate immediate effector functions. Tfh cells provide help to DENV-specific germinal center B cells yellow and are essential for optimal germinal center reactions, thus promoting the generation of high-affinity antibodies, memory B cells, and long-lived plasma cells.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. National Center for Biotechnology Information , U. Front Immunol. Published online Sep 4. Author information Article notes Copyright and License information Disclaimer.

This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology. Received Mar 1; Accepted Aug The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.

No use, distribution or reproduction is permitted which does not comply with these terms. Its antigens consist of chimeric viruses in which genomic sequences encoding the pre-membrane prM protein and the envelope glycoprotein E of the 17D strain of Yellow fever virus YFV were replaced by those of each of the four serotypes of DENV.

The levels of presence and absence of epitopes that are target for protective immune response in the three main anti-dengue virus vaccines are shown here. We performed a search for anti-dengue vaccine formulations both, in clinical trial records ClinicalTrials.

The search filters used at ClinicalTrials. Pubmed searches were performed using keywords: dengue, vaccine, clinical trial. The dataset was used for the further analyses. The DENV polyprotein sequences dataset was built by applying the same filters for Flavivirus previously described Sequences were retrieved until January Additionally, we carried out a bibliography search for the studies about the protective anti-dengue response induced by T lymphocyte epitopes that were experimentally characterized.

We also selected only studies in which at least two different methods were used to validate the secretion of pro-inflammatory cytokines or cytotoxic activity.

A total of epitopes were retrieved from five articles see Supplementary Table 3. However, eight of them were found more than once and, therefore, repeated epitopes were removed from the dataset for the further analyses. Briefly, we computed the conservation of B cell epitopes retrieved from the IEDB database and T cell epitopes characterized and reported in the literature. Epitopes conserved in vaccines were mapped in a 3D envelope glycoprotein model, as previously described Statistical analysis was performed to compare the results of the epitope conservation scores that were present in at least one of the three vaccines.

Scores were defined based on the levels of conservation of epitopes present in vaccines in circulating viruses. This method is shown in details in Supplemental Material. Comparisons were performed regarding location of epitopes in specific viral proteins and percentage of conservation in circulating viruses clinical isolates.

The Kruskal-Wallis one-way test was used to verify whether the differences in the medians of the set of epitopes present in each protein were significant.

This statistical analysis was only necessary for the comparison of the conservation values among the T cell epitopes. We did not compare conservation regarding B cell epitopes because they do not vary in relevant levels. The vaccine constructs were assembled in the ApE-A plasmid Editor. Figure 1 Schematic representation of genetic constructions of vaccine antigens.

Known accession numbers are shown. NS, non-structural proteins. This alteration contributes to viral attenuation, as previously described TAK is a tetravalent vaccine. In order to build the chimeras, we used the nucleotide sequence from PDKV, according to the accession number in Figure 1G. Finally, the nucleotide sequences of vaccine viruses were translated and deposited in a sequence dataset in FASTA format see Supplementary Data.

It is important to highlight that such epitopes are target for neutralizing antibodies which were previously reported. We evaluated the conservation of these 50 epitopes in vaccine antigens from the three manufacturers. However, we did not find relevant differences in the number of epitopes conserved among the vaccine antigens studied here see Table 1. It is important to highlight that all of these conserved epitopes are discontinuous.

In addition, most of them are located in the envelope glycoprotein and only one of them is located in prM. Moreover, our analyses showed that circulating DENV2 contain the highest number B cell epitopes targeted by neutralizing antibodies see Supplementary Table 4.

Table 1 B cell epitopes which are target for neutralizing antibodies and are conserved in the main dengue vaccines. We identified the presence of a diverse distribution, with epitopes inserted in all regions of the envelope glycoprotein: DI, shown in red; DII, shown in yellow, DIII, shown in blue and the fusion loop shown in green see Figure 2A.

Epitopes with ID , , and are highlighted and contain all their residues fully inserted in the fusion loop region. Residues shared by these two epitopes out of the fusion loop are shown in cyan. The Epitopes with ID , , and contain amino acids in the DIII, and also share common amino acids in the stem region, which is not present in the model used in this study Figure 2C.

Finally, only one epitope is located in the prM ID It is conserved in the three vaccines, as shown in Table 1. It is important to highlight that some of the epitopes presented here are conserved in both vaccines and circulating viruses see Supplementary Table 4. These results suggest that the three vaccines antigens analyzed in this study contain most of B cell epitopes targeted by neutralizing antibodies.

Figure 2 Location B cell epitopes that are the target for neutralizing antibodies and conserved in dengue vaccines on the 3D structure of envelope glycoprotein dimers. A 3D structure of envelope glycoprotein dimer. B Location of the 5 epitopes that share residues in the fusion loop, shown in green.

Three of these epitopes contain all of their residues located in the fusion loop , e The three last epitopes have residues located at the stem region; which was not represented in this 3D model. D Representation of the epitope with ID , with residues shown in salmon. All residues are located at DII. The conservation analysis revealed that 29 out of T cell epitopes were not conserved in the three vaccine antigens, whereas two of them were not preserved in either vaccine or circulating viruses Supplementary Table 5.

However, two and five of them were found in prM and NS5, respectively. A score-based scale associated with conservation percentages of epitopes obtained from circulating viruses Supplementary Table 6 was used to statistically compare conservations of epitopes in the three studied dengue vaccines.

Table 2 Results of the medians of the epitope set scores and the p-values derived from comparative analyzes between vaccines. The epitopes were evaluated regarding their coverage in the world population.