Dengue is one of the most globally serious vector-borne infectious diseases in tropical and subtropical areas for which there are currently no effective vaccines. The most highly conserved flavivirus protein, NS5, is an indispensable target of CD8+ T-cells, making it an ideal vaccine design target. Using the Immune Epitope Database (IEDB), CD8+ T-cell epitopes of the dengue virus (DENV) NS5 protein were predicted by genotypic frequency of the HLA-A,-B, and-C alleles in Chinese population. Antigenicity scores of all predicted epitopes were analyzed using VaxiJen v2.0. The IEDB analysis revealed that 116 antigenic epitopes for HLA-A (21),-B (53), and-C (42) had high affinity for HLA molecules. Of them, 14 had 90.97–99.35% conversancy among the four serotypes. Moreover, five candidate epitopes, including 200NS5210 (94.84%, A*11:01), 515NS5525 (98.71%, A*24:02), 225NS5232 (99.35%, A*33:03), 516NS5523 (98.71%, A*33:03), and 284NS5291 (98.06%, A*33:03), were presented by HLA-A. Four candidate epitopes, including 234NS5241 (96.77%, B*13:01), 92NS599 (98.06%, B*15:01, B*15:02, and B*46:01), 262NS5269 (92.90%, B*38:02), and 538NS5547 (90.97%, B*51:01), were presented by HLA-B. Another 9 candidate epitopes, including 514NS5522 (98.71%, C*01:02), 514NS5524 (98.71%, C*01:02 and C*14:02), 92NS599 (98.06%, C*03:02 and C*15:02), 362NS5369 (44.84%, C*03:04 and C*08:01), 225NS5232 (99.35%, C*04:01), 234NS5241(96.77%, C*04:01), 361NS5369 (94.84%, C*04:01), 515NS5522 (98.71%, C*14:02), 515NS5524 (98.71%, C*14:02), were presented by HLA-C. Further data showed that the four-epitope combination of 92NS599 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02), 200NS5210 (A*11:01), 362NS5369 (C*03:04, C*08:01), and 514NS5524 (C*01:02, C*14:02) could vaccinate >90% of individuals in China. Further in vivo study of our inferred novel epitopes will be needed for a T-cell epitope-based universal vaccine development that may prevent all four China-endemic DENV serotypes.
Citation: Shi J, Sun J, Wu M, Hu N, Li J, Li Y, et al. (2015) Inferring Protective CD8+ T-Cell Epitopes for NS5 Protein of Four Serotypes of Dengue Virus Chinese Isolates Based on HLA-A, -B and -C Allelic Distribution: Implications for Epitope-Based Universal Vaccine Design. PLoS ONE 10(9): e0138729. https://doi.org/10.1371/journal.pone.0138729
Editor: Lisa F.P. Ng, Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), SINGAPORE
Received: July 5, 2015; Accepted: September 2, 2015; Published: September 18, 2015
Copyright: © 2015 Shi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This research was supported by a grant from the Applied and Fundamental Research program of Yunnan Province (Grant No. 2013FA025), which was received by YZH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Dengue virus (DENV) can cause dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), globally important mosquito-borne diseases [1, 2]. These are among the most serious epidemic arbovirus diseases and endemic in tropical and subtropical regions of the word. The causative viruses are members of the genus Flavivirus within the family Flaviviridae and can be grouped into four antigenically distinct serotypes (DENV1-4) that share 67–75% sequence homology [3, 4]. DENV is transmitted to humans through the bites of infected Aedes aegypti and Aedes albopictus mosquitoes. Nearly half of the world’s population is under risk of contracting dengue. It is estimated that up to 390 million infections occur annually worldwide with approximately 96 million symptomatic cases . Despite more than 60 years of effort, no licensed vaccine is currently available. Thus, the search for a safe and effective vaccine is growing more imperative.
Dengue is hyperendemic and has become a serious public health concern in China. The first outbreak of dengue was reported in Guangdong Province of China in 1978 [6, 7]. Since then, annual DENV epidemics have occurred, followed by a dengue epidemic in Guangxi, Fujian, Zhejiang, and other areas of China. In 2014, the most serious dengue epidemic in history occurred in Guangdong province of China with a total of 48,162 infected individuals . This outbreak is considered an imported epidemic from neighboring Southeast Asian countries [9, 10]. In recent years, the scope of the epidemic is further expanding from the coastal city of China to inland cities. In 2013, an outbreak of DENV occurred in Yunnan province of China with more than 2,000 infected individuals . A safe and effective dengue vaccine is urgent need in China.
CD8+ T-cell-mediated immunity plays an important role for eliminating intracellular pathogens. Thus, eliciting robust CD8+ T-cell immunity is the basis for many vaccines under development. Although DENV-specific CD8+ T-cell responses have been extensively studied, the vast majority of studies focused on immunopathogenic role of T-cells during DENV infection [12–14]. The viewpoint from these studies is that serotype cross-reactive CD8+ T-cells may contribute to the immunopathogenesis of DHF/DSS. Thus, the vast majority of dengue vaccine candidates are designed to produce protective neutralizing antibodies with less regard for cellular immune responses. However, direct evidence linking T-cells to increased viremia or DENV-related pathology has not been demonstrated. Notably, recent extensive studies have demonstrated a protective role of CD4+ and CD8+ T-cells against homologous or heterotypic DENV infection in murine models [15–20]. Specifically, these studies demonstrated that CD8+ T-cells can control viral replication , prevent antibody-dependent enhancement (ADE) of infection , and DENV-induced CNS disease .
These findings are consistent with the murine model data of a recent study supporting the concept of a protective role of T-cells against DENV infection in humans. The results of this study showed that the secondary DENV infection in humans was not significantly associated with disease severity . Further, another recent study provided the first comprehensive map of the CD8+ T-cell response to DENV in humans and support a HLA-linked protective but not pathogenic role for CD8+ T-cells against DENV infection in humans . Collectively, these findings strongly imply a protective role for CD8+ T-cells against severe DENV disease in humans. Based on these studies, it is inferred that the lack of induction of a robust DENV-specific T-cell response may be a reason for the results of a recent efficacy trial of the most advanced dengue vaccine candidate, a tetravalent live-attenuated chimeric vaccine (CYD) based on the 17D-attenuated yellow fever virus backbone that showed only partial protection despite the induction of DENV-specific neutralizing antibody to each serotype in most subjects . This means that the roles of T-cells in the context of DENV vaccination should not be ignored, and it raises the possibility that T-cell responses against all DENV serotypes might be beneficial or even required for vaccine protective efficacy. The advent of a T-cell epitope–based vaccine may offer an alternative that avoids ADE. Considering the important role of serotype-specific CD8+ T-cells in controlling DENV infection, a novel strategy for developing prophylactic and therapeutic CD8+ T-cell epitope-based vaccines is needed. Therefore, a T-cell epitope-based universal vaccine that induces a broad dengue-specific, multifunctional, and cross-reactive CD8+ T-cell responses among all four DENV serotypes may be a more promising strategy against DENV infections.
The DENV genome consists of a single-stranded RNA of 10.7 kb in length. The open reading frame codes three structural proteins [capsid (C) protein, preM protein, and envelope (E) protein] and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) [24,25]. It has been shown that CD8+ T cells preferentially target the NS3 and NS5 proteins, while CD4+ T cells preferentially target the E, C, and NS1 proteins . Notably, NS5 is the largest and the most highly conserved protein encoded by the DENV genome, with approximately 67–82% amino acid sequence identity among the four DENV serotypes . Thus, NS5 proteins could be used as a promising target in the design of a T-cell epitope-based vaccine to induce DENV-specific protective T-cell responses.
A necessary condition for a peptide to be a CD8+ T-cell epitope is that it binds to human leukocyte antigen (HLA) molecules. However, HLA molecules are extremely polymorphic with several thousand variants and can bind distinct sets of peptides . Each HLA variant is expressed at vastly variable frequencies in different ethnic groups and geographic regions . This means that it appears that an extremely large and impractical number of peptides would have to be selected to enable the development of a broadly protective multi-epitope vaccine. A large number of studies focused on predicting epitopes from the E, prM, NS1, NS3, or NS5 proteins allowed the identification of T-cell epitopes in DENV [30–36]. However, the CD8+ T-cell epitopes of the NS5 protein of DENV Chinese isolates linked with the class I HLA allele in Chinese population have been poorly revealed. Therefore, the identification of CD8+ T-cell epitopes that can induce protective DENV-specific T-cell responses by a feasible immunoinformatics approach is critical and urgent for the development of a T-cell epitope-based vaccine.
In this study, based on the distribution characteristics of HLA class I alleles in Chinese population, we identified putative CD8+ T-cell epitopes of NS5 protein of Chinese DENV isolates using various immunoinformatics approaches. Our results provide putative protective CD8+ T-cell epitope candidates or their combination for the development of a T-cell epitope-based universal vaccine to effectively prevent all four DENV serotypes that are endemic in China.
Materials and Methods
Retrieving the protein sequences
The sequences of the NS5 protein from 155 Chinese isolates belong to all four serotypes of dengue virus (DENV-1, DENV-2, DENV-3 and DENV-4) were retrieved from the National Center for Biotechnology Information (NCBI) protein database (http://www.ncbi.nlm.nih.gov/protein/). The sequence of DENV-2 NS5 protein (accession number: KC131142.1) was used as an input for various bioinformatics tools for epitope prediction, antigenicity analysis and conservation analysis.
HLA genotypic frequency retrieval
To improve population coverage of the CD8+ T-cell epitopes, it is important to screen epitopes restricted by highly prevalent HLA alleles. Genotypic frequencies of the HLA class I alleles that include HLA-A,-B and-C loci in Chinese population were retrieval from the major histocompatibility complex database (dbMHC) (http://www.ncbi.nlm.nih.gov/projects/gv/mhc/main.fcgi?cmd=init). For a broad coverage, HLA class I alleles with genotypic frequency >3% in Chinese population were selected for CD8+ T-cell epitope prediction. This parameter setting covers the highly prevalent HLA-A,-B, and-C alleles found in Chinese population.
CD8+ T-cell epitope is the minimal amino acid sequence required for CD8+ T-cells activation and recognition by immune system receptors. Since the affinity of an epitope binds to the HLA molecule plays a vital role in determining its immunogenicity. Hence, high affinity between epitopes and HLA molecules tends to be associated with higher immune responsiveness. The prediction of CD8+ T-cell epitopes that interact with different HLA class I alleles were performed using the IEDB analysis resource (http://tools.immuneepitope.org/mhci/) Consensus tool , which combines predictions from ANN, aka NetMHC 3.4 , SMM  and Comblib , if any corresponding predictor is available for the molecule. Otherwise, NetMHCpan is used. This choice was motivated by the expected predictive performance of the methods in decreasing order: Consensus > ANN > SMM > NetMHCpan > CombLib. For the IEDB-recommended method, a low percentile indicated strong binding affinity to HLA molecules. The threshold of percentile rank was set at 1 in this study . Based on the representative length of peptides that bind to HLA molecules, peptide lengths of 8–11 amino acids were selected for the prediction of epitope-based peptides in this study.
Antigenicity is a key characteristic of the epitope that is recognized by immune system cells and/or antibodies. Thus, the antigenicity of the predicted epitopes is one of the most important criteria for epitope-based vaccine assessment. Since some of the predicted epitopes may lose antigenicity when analyzed, to ensure that the predicted epitopes could serve as a good CD8+ T-cell epitope, all of the epitopes were screened to assess their antigenicity. VaxiJen v2.0 (http://www.ddg-pharmfac.net/vaxijen/VaxiJen), an online web server used to predict the effective antigens and subunit vaccines, was used to identify and reevaluate T-cell epitope antigenicity. The predicted epitopes were uploaded in a plain sequence format and the virus was chosen as the target organism. The threshold level of an antigen was set at 0.5. The Vaxijen server performed well with 87% accuracy at a threshold of 0.5 antigenic score for viruses . VaxiJen v2.0 allows antigen classification based on the physicochemical properties of proteins without recourse to sequence alignment. Finally, the epitopes with an antigenic score > 0.5 were selected as antigenic for the conservancy analysis.
Conservancy and population coverage analysis
To obtain the universal T-cell epitopes of four DENV serotype variants, the conservancy of candidate epitopes should be considered prior to other criteria, even population coverage rate. As a universal T-cell epitope, it should be highly conserved in all viral variants. Hence, to determine the conservation level of the predicted epitopes among the NS5 protein sequences of the different DENV strains, the predicted epitopes were analyzed for their conservancy using the IEDB epitope conservancy tool (http://tools.immuneepitope.org/tools/conservancy/iedb_input) with a sequence identity threshold of 100%. The conservancy level of each potential epitope was calculated by seeking identities in all NS5 protein sequences of the four DENV serotype variants retrieved from the NCBI protein database. The epitopes that were 100% conserved in >90% of the sequences analyzed in four serotypes were selected as candidate epitopes. These highly conserved epitopes were selected and used to determine the population coverage by the IEDB population coverage calculation tool (http://tools.immuneepitope.org/tools/population/iedb_input). Finally, all of the selected epitopes were analyzed for similarity with human proteome using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) to verify that they would not trigger autoimmunity.
Results and Discussion
Retrial of NS5 protein sequences of four DENV serotypes
The NS5 proteins sequence of all four DENV serotypes circulating in China were retrieved in FASTA format from the NCBI protein database. A total of 155 sequences of the NS5 protein of Chinese isolates of the four DENV serotypes was obtained (S1 File, S2 File, S3 File and S4 File) and used for the further epitope analysis.
HLA class I alleles analysis
Since specific HLA alleles are expressed at variable frequencies in different ethnic groups and different geographic regions. Therefore, HLA allele frequencies prevalent in dengue hyperendemic areas must be considered in vaccine design. Here we focused on DENV-specific T-cell epitopes that are associated with the highly prevalent HLA alleles in a Chinese population. To this end, the genotypic frequency of the highly prevalent HLA class I alleles found in this Chinese population (>3%) that include HLA-A,-B, and-C loci were obtained from the dbMHC database. As a result, seven HLA-A alleles (A*02:01, A*02:03, A*02:06, A*02:07, A*11:01, A*24:02, and A*33:03), eight HLA-B alleles (B*13:01, B*15:01, B*15:02, B*38:02, B*40:01, B*46:01, B*51:01, and B*58:01), and eight HLA-C alleles (C*01:02, C*03:02, C*03:03, C*03:04, C*04:01, C*08:01, C*14:02, and C*15:02) were obtained (Table 1).
Prediction and antigenic analysis of CD8+ T-cell epitopes
CD8+ T-cell responses play a substantial role in eliminating DENV infected cells that cannot be managed by antibody responses. An effective vaccine that can provide protection against dengue virus infection requires robust, broad, and multi-functional CD8+ T-cell responses. Therefore, the CD8+ T-cell epitopes that bind to different HLA class I alleles with varying affinities must first be identified. Here a large number of the antigenic epitopes with a high binding affinity score of <1 percentile and an antigenicity score > 0.5 were obtained from NS5 proteins of DENV Chinese isolates against HLA-A,-B, and-C alleles. Most epitopes bind with high affinity to single HLA-A,-B, or-C molecules. As a consequence, a total of 21 antigenic epitopes were obtained against the seven alleles of the HLA-A loci (Table 2). A total of 53 antigenic epitopes were obtained against the eight alleles of the HLA-B loci (Table 3), while 42 antigenic epitopes were obtained against the eight alleles of the HLA-C loci (Table 4). Surprisingly, none of the epitopes bind to HLA-A*02:07. Notably, some epitopes, like 92AMTDTTPF99 (B*15:01, B*15:02, B*46:01, C*03:02, and C*15:02), 515MYFHRRDLRL524 (A*24:02 and C*14:02), 225WYMWLGAR232 (A*33:03 and C*04:01), 234LEFEALGF241 (B*13:01 and C*04:01), 514LMYFHRRDLRL524 (C*01:02 and C*14:02), and 362FTNMEAQL369 (C*03:04 and C*08:01) can be presented by multiple HLA molecules, suggesting that they can cover a broader population and may be better epitope vaccine candidates. Further, the potential of the epitope and HLA binding is essential in the assessment of the immunogenic potential of epitopes. Hence, as a ligand of the HLA-A molecule, epitopes 225WYMWLGAR233 and 224IWYMWLGARF233 presented by HLA-A*24:02 were the best binders based on their 0.2 percentile. As ligands of HLA-B molecules, epitopes 89TQMAMTDTTPF99 and 530CSAVPSHW537, which were presented by HLA-B*15:01 and HLA-B*58:01, respectively, were the best binders based on their 0.1 percentile. Likewise, as a ligand of HLA-C molecule, epitope 91MAMTDTTPF99 presented by HLA-C*03:02 was the best binder based on its 0.1 percentile. Additionally, HLA-A*24:02 has the highest number of binding epitopes (9/21), followed by A*33:03 (6/21) in the HLA-A allele. HLA-B*13:01 and HLA-B*58:01 presented the same most frequent epitopes (13/53), followed by B*15:01 (12/53), B*46:01 (12/53), B*38:02 (11/53), and B*15:02 (9/53) in the HLA-B alleles. Moreover, HLA-C*04:01 presented the most frequent epitopes (17/42), followed by C*14:02 (13/42), C*01:02 (8/42), C*03:02 (8/42), and C*15:02 (7/42) in the HLA-C alleles. These calculations were made on the basis of HLA genotypic frequencies assuming non-linkage disequilibrium between HLA loci. Overall, these results provide in silico insight for class I HLA allele-restricted CD8+ T-cell epitopes against the NS5 protein of DENV in Chinese population.
Conservancy and population coverage of CD8+ T-cell epitopes
As an effective vaccine formulation, the epitope-based universal vaccine must include highly conserved CD8+ T-cell epitopes among all DENV serotypes to induce cross-reactive T-cell responses based on the fact that the conserved epitope candidates are more likely to confer cross-protection between pathogen variants. Here, conservancy analysis revealed a total of 14 highly conserved epitopes with ≥90% protein sequence matching in a total of 155 NS5 protein sequences from four DENV serotypes of Chinese isolates. For the HLA-A allele, five of the 21 epitopes were conserved with ≥90% conservancy (Table 2), including 200CVYNMMGKREK210 (94.84%, A*11:01), 515MYFHRRDLRL524 (98.71%, A*24:02), 225WYMWLGAR232 (99.35%, A*33:03), 516YFHRRDLR523 (98.71%, A*33:03), and 284DTAGWDTR291(98.06%, A*33:03). For the HLA-B allele, four of the 53 epitopes were conserved with ≥90% conservancy (Table 3), including 234LEFEALGF241 (96.77%, B*13:01), 92AMTDTTPF99 (98.06%, B*15:01, B*15:02 and B*46:01), 262LHKLGYIL269 (92.90%, B*38:02), and 538VPTSRTTWSI547 (90.97%, B*51:01). For the HLA-C allele, nine of the 42 epitopes were conserved with ≥90% conservancy (Table 4), including 514LMYFHRRDL522 (98.71%, C*01:02), 514LMYFHRRDLRL524 (98.71%, C*14:02 and C*01:02), 92AMTDTTPF99 (98.06%, C*03:02 and C*15:02), 362FTNMEAQL369 (94.84%, C*03:04 and C*08:01), 225WYMWLGAR232 (99.35%, C*04:01), 234LEFEALGF241(96.77%, C*04:01), 361TFTNMEAQL369 (94.84%, C*04:01), 515MYFHRRDL522 (98.71%, C*14:02), and 515MYFHRRDLRL524 (98.71%, C*14:02).
Since the HLA allele frequencies vary among populations due to different genetic backgrounds, to design an effective vaccine, we should consider the candidate epitopes that specifically bind with the prevalent HLA molecules in the target population where the vaccine will be employed. Therefore, here we examined the population coverage of the proposed epitope vaccine candidate in a Chinese population. The results showed that the epitope 92AMTDTTPF99 (B*15:01, B*15:02, B*46:01, C*03:02, and C*15:02) has the highest percentage of population coverage (47.16%), followed by 200CVYNMMGKREK210 (A*11:01, 43.48%), 362FTNMEAQL369 (C*03:04 and C*08:01, 36.60%), 514LMYFHRRDLRL524 (C*01:02 and C*14:02, 33.53%), 515MYFHRRDLRL524 (A*24:02 and C*14:02, 28.69%), and 514LMYFHRRDL522 (C*01:02, 27.68%) in China (Table 5). It is worth noting that the combination of the epitopes 92AMTDTTPF99 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02), 200CVYNMMGKREK210 (A*11:01), 362FTNMEAQL369 (C*03:04 and C*08:01), and 514LMYFHRRDLRL524 (C*01:02 and C*14:02) could vaccinate >90% of the Chinese population, suggesting that the four epitopes are better candidates for a multiple T-cell epitope-based vaccine. These highly conserved HLA restricted epitopes with acceptable population coverage could be putative epitope vaccine candidates in their combinations to elicit DENV-specific T-cell responses. Finally, to avoid the autoimmune response, all of the predicted class I HLA-binding antigenic epitopes were analyzed for their homology with human proteome, but no epitope was homologous with human proteome. Based on these results, we proposed that the combination of these highly conserved epitopes could be as universal CD8+ T-cell epitope vaccine candidates to induce DENV-specific T-cell responses against four DENV serotypes that are endemic in China.
HLA-restricted epitopes for prophylactic or therapeutic vaccines against infectious diseases to induce a T-cell response that eliminates infected cells is a promising vaccine strategy. In this study, we identified 14 universal CD8+ T-cell epitope candidates using immunoinformatic approach, and they are highly conserved among all four DENV serotypes that are endemic in China. The combination of four epitopes, including 92AMTDTTPF99 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02), 200CVYNMMGKREK210 (A*11:01), 362FTNMEAQL369 (C*03:04 and C*08:01), and 514LMYFHRRDLRL524 (C*01:02 and C*14:02), could vaccinate >90% of individuals in China. These epitopes are valuable T-cell epitope-based vaccine candidates for the development of a universal dengue vaccine that is capable of eliciting specific and robust protective T-cell responses against four DENV serotype variants. In conclusion, our study highlights that it is possible to design an epitope-based universal vaccine against all four DENV serotypes based on protective CD8+ T-cell-mediated cellular immune responses.
S1 File. All available NS5 protein sequences of dengue virus serotype 1 Chinese isolates.
S2 File. All available NS5 protein sequences of dengue virus serotype 2 Chinese isolates.
S3 File. All available NS5 protein sequences of dengue virus serotype 3 Chinese isolates.
Conceived and designed the experiments: JDS YZH. Performed the experiments: JDS JS MNW NZH JFL YHL HXW. Analyzed the data: JDS. Contributed reagents/materials/analysis tools: JS NZH. Wrote the paper: JDS.
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