Prediction of T cell epitopes for the utility of vaccine development from structural proteins of dengue virus variants using in silico methods

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1 Indian Journal of Biotechnology Vol 8, April 2009, pp Prediction of T cell epitopes for the utility of vaccine development from structural proteins of dengue virus variants using in silico methods Pallavi Somvanshi * and P K Seth Bioinformatics Centre, Biotech Park, Sector-G, Jankipuram, Lucknow , India Received 1 February 2008; revised 29 September 2008; accepted 5 December 2008 The spread of dengue virus, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) has increased significantly in the past two decades and thus has been a major concern of public health globally. Dengue virus infection can lead to a wide range of manifestations in the form of undifferentiated fever, classic dengue fever, DHF with plasma leakage, which leads to hypovelmic shock DSS. A new strategy for developing prophylactic and therapeutic application of pathogenspecific immunity was provided from epitope-based vaccines; it is a critical requirement for the identification and selection of T cell epitopes that act as vaccine target. Immunoinformatics serves as a valuable tool to screen and select antigenic peptide sequences as potential T cell epitopes for binding affinity with HLA alleles. We studied dengue variants conserved epitopes in three structural proteins, capsid, envelope and precursor membrane, which recognize some highest binding affinity HLA. A total of 45 promiscuous nanomer candidate epitopes for HTL are recognized against MHC Class II and 28 promiscuous epitopes are recognized against CTL for MHC class I. This computational prediction analysis will improve our understanding of T cell immune response and help in identifying the antigenic peptide for formulation of antigen based diagnostic kit and peptide based subunit vaccine design against dengue virus. Keywords: Dengue, epitopes, HLA, vaccine, in silico, structural proteins Introduction Dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), have become the major public health problems globally; particularly in the tropical and subtropical countries 1,2. Dengue, a mosquito borne disease caused by the four serotypes of dengue virus, is characterized by mild symptoms to hemorrhagic manifestations and shock syndrome 3. Dengue virus is the most important arthropod-borne viral infection transmitted primarily by the female Aedes aegypti mosqutoes. Other Aedes species have also been implicated in the transmission of the infection 4.. Dengue outbreaks and epidemics continue to pose a public health problem in most tropical and subtropical regions of the world 5.The dengue virus infection has a high mortality rate, approximately 50 to 100 million dengue virus infections and up to 250,000 dengue hemorrhagic fever cases occur every year worldwide 2. The four dengue virus serotypes (DENV 1-4) constitute the dengue virus complex within the *Author for correspondence: Tel: ; Fax: psomvanshi@gmail.com Flavivirus genus of the Flaviviridae family 6. The virus genome contains a positive-strand RNA with one open reading frame coding for polyprotein. The polyprotein is processed to produce three structural proteins, the capsid (C), precursor membrane (prm), and envelope (E), besides seven non-structural proteins, designated as NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 7. A single polypeptide is cotranslationally processed by viral and cellular proteases generating only three structural proteins, capsid (C; molecular mass, kda) which binds to the positive strand genomic RNA forming the nucleocapsid, and two lipid associated membrane (M; molecular mass, 7-8 kda) and large membrane protein, which is also called envelope glycoprotein (E; molecular mass, kda). The envelope glycoprotein is the major virion antigen responsible for virus neutralization by specific antibodies and for several important antigenic properties; it encodes the important biological functions of attachment to cellular receptor, membrane fusion and elicitation of virus neutralizing antibodies, and at least seven non-structural proteins. The function of envelope protein is to control the receptor binding and fusion with the host cell 8. The dimeric capsid or core protein represents the least conserved structural protein. Its main function is

2 194 INDIAN J BIOTECHNOL, APRIL 2009 packaging of viral RNA, forming the nucleocapsid, binding of viral RNA, mediating viral assembly, regulation of the viral lifecycle 9 together with E, C and the viral genome 1. The premembrane protein functions as the chaperone for folding and assembly of the E protein. The E protein is responsible for viral attachment to the putative cell surface receptor(s), fusion with the endosomal membranes upon entry, and mediating protective immune responses in the infected host 10. Despite 6 decades of research, neither a safe and effective dengue vaccine has been developed nor a specific, short-term preventive measure available. Currently, prevention of dengue is carried out by mosquito vector control, which is rather ineffective. Unfortunately, many countries affected by DEN viruses have very limited financial resources for healthcare and economic burden of DEN disease is considerable. However, development of a vaccine against such virus immunity requires high bio-safety level containment facilities and it is also difficult in some cases to obtain high virus yields in cell culture because of high virus pathogenicity. Several approaches have been used in reverse genetics techniques, DNA vaccination and the use of related non-pathogenic dengue virus with and without different adjuvants. Computational immunology methods dramatically reduce the time and efforts (up to 95%) involved in the screening of potential epitopes 11. Significant efforts have been made in the last few years as several groups devoted their research towards the development of procedures and algorithms that allow more effective and accurate prediction of MHC binding affinity. Many web based tools are publicly available for predicting T-cell epitopes from the protein sequence and have been widely used by experimental researchers without expert knowledge of bioinformatics 12. The present study was conducted to predict and identify the promiscuous epitope peptide that binds to HLA molecules computationally. It might be useful for peptidebased diagnosis of dengue. We have also found conserved new epitopes in dengue protein that may be used as diagnostic reagents and safe subunit vaccines against the dengue fever. Materials and Methods A set of publicly available bioinformatics algorithms and computational tools were used to screen and select antigen sequences as promiscuous cellular immune response in the dengue virus variants. The complete protein structural sequences of dengue viruses I, II, III and IV were taken from NCBI Genbank. The open reading frame (ORF), restriction site, expected molecular weight and isoelectric point (pi) values were analyzed with Generunner and DNAstar (Lasergene). Prediction programs, ProPred and ProPred I, were used to find the conserved and antigenic domains of structural proteins of dengue virus variants. MHC molecules are cell surface glycoproteins, which take active part in host immune reactions. The counterparts in human are known as human leukocyte antigens (HLA). The MHC molecules are coded by three classes of genes. Class I and II gene products are directly associated with immune reactions whereas Class III gene products play an indirect role. Class I genes encode the principle subunits of MHC I glycoproteins called H2-K, H2-d, H2-l in mice and HLA- A, B and C in humans. Proteins encoded by these genes are present virtually on all nucleated cells. Class II genes encode cell surface glycoproteins which are structurally very similar to MHC Class I molecules. These molecules are expressed only on antigen presenting cells (APC) together with antigenic fragments, the Class II proteins form epitopes that are recognized by T-helper cells (CD4+). Hence, MHC Class II proteins are critically involved in response to nearly all antigens. ProPred allows to predict MHC class II binding peptides (HTL epitopes) for 51 alleles and ProPred I to predict MHC class I binding peptides (CTL epitopes) for 47 alleles. The highest scorer helper T lymphocytes (HTL) are selected at the threshold level (1%) for ProPred and in the case of ProPred I cytotoxic T lymphocytes (CTL), s at the threshold level (4%) with proteasome filter 5% and showed the binding affinity with more than 4 MHC alleles (promiscuous) which do not overlap in the amino acid sequence. Results and Discussion In the present study, we analyzed dengue virus variant structural region of the polyprotein, which consists of 3 structural subunit proteins like envelope, capsid and premembrane. The nature of these proteins was theoretically analyzed with computational software. The molecular weight and pi of all these three proteins deduced with the use of

3 SOMVANSHI & SETH: PREDICTION OF T CELL EPITOPES 195 immunoinformatic software are given in Table 1. The theoretical pi value of envelope proteins, ranged from 6.83 to 7.52, capsid to and the premembrane 5.59 to 6.97, respectively. The pi values of all these structural proteins indicated the stability of protein at that particular pi. Based on these results several epitopes were predicted and identified that were present in these structural proteins. We have also found some highly conserved antigenic epitopes present in the protein, which may be useful for the diagnosis of different variants of dengue. However, using the variant Proteins from different sources of virus (Dengue 1) (Dengue 2) (Dengue 3) (Dengue 4) specific antigenic epitopes may do dengue variant specific diagnostic. We have identified several variant specific epitopes from envelope, capsid and precursor membrane proteins. Four different dengue viruses I, II, III and IV, were taken specifically. Each variant had three major structural parts of proteins. Therefore, total 12 structural proteins were analyzed through immuno-informatics. The predicted epitopes and MHC alleles were used in the ProPred and ProPred1 for envelope, capsid and premembrane proteins of dengue virus variants (Tables 2, 3 and 4). Some scholars speculate that CTL mediated cellular Table 1 Deduced physico-chemical properties of dengue virus proteins Protein designation Accession no. s MW kda pi value protein Dengue virus type 1 ABQ Dengue virus type 2 ABK Dengue virus type 3 AAQ Dengue virus type 4 AAU protein Dengue virus type 1 NP_ Dengue virus type 2 ABK Dengue virus type 3 AAP Dengue virus type 4 NP_ Premembrane protein Dengue virus type 1 AAL Dengue virus type 2 CAA Dengue virus type 3 AAL Dengue virus type 4 ABO Table 2 Predicted epitopes for dengue virus variants of envelope proteins using immunoinformatics tools Predicted HTL ProPred Number of MHC Class II binding alleles Predicted CTL ProPred1 LKMDKLTLK WLVHKQWFL WFLDLPLPW RGWGNGCGL IVQYENLKY LGLNSRSTS LKMDKLTLK LRMDKLQLK VLLQMESKV MRGAKRMAI WLVHRQWFL MKILIGVVI LVLVGIVTL IGMNSRSTS LVTCAKFQC GLFGKGSLV LKMDKLELK LKYTVIITV IGIGDKALK WLVHKQWFL IGLNSKNTS VLNSLGKMV LYLGAVVQA WIMKIGIGV YRGAKRMAI WMVRILIGL MVRILIGLL VVTCAKFSC YIVIGVGDS LFLGFTVQA Number of MHC Class I binding alleles

4 196 INDIAN J BIOTECHNOL, APRIL 2009 Proteins from different sources of virus Table 3 Predicted epitopes for dengue virus variants of capsid proteins using immunoinformatics tools Predicted HTL ProPred Number of MHC Class II binding alleles immune response probably plays an important role in viral clearance 13,14. CD8+ CTL interact through their polymorphic T cell receptor with HLA class I molecules containing endogenously synthesized peptides of 9-11 on the surface of infected cells. The presence of allele-specific amino acid motifs has been demonstrated by sequencing of peptides eluted from MHC molecules (Tables 2,3 & 4). The promiscuous peptide recognized by the T cells mediated immunity assay may have the potential in developing peptide based vaccine and diagnostic reagent against dengue viruses. An important requirement for vaccine candidate was their promiscuous presentation to Th1 cell mediation of protective immunity against dengue virus. The identification of promiscuous Th1 cell epitopes of three structural protein antigens of dengue virus Predicted CTL ProPred1 LVMAFIAFL LVMAFIAFL 77 5 (Dengue 1) LRGFKKEIS KEISNMLNI LNIMNRRKR VQQLTKRFS ALVAFLRFL 66 4 (Dengue 2) LRFLTIPPT LVMAFIAFL LVMAFIAFL 55 5 LRFLAIPPT FKKEISNML 93 4 (Dengue 3) IINKRKKTS ITFLRVLSI 49 7 MVLAFITFL 45 4 (Dengue 4) IKILIGFRK 76 3 FSGKGPLRM 37 3 TPQGLVKRF 24 4 Proteins from different sources of virus Table 4 Predicted epitopes for dengue virus variants of premembrane proteins using immunoinformatics tools Predicted HTL ProPred Number of MHC Class II binding alleles Predicted CTL ProPred1 Number of MHC Class I binding alleles Number of MHC Class I binding alleles WVTYGTCNQ LLFKTSAGV 54 4 Premembrane LAPHVGMGL YKCPRITEA 82 4 (Dengue 1) FKTASGINM 55 3 ILLTAVAPS LLFKTGDGV 23 4 Premembrane (Dengue 2) FTIMAAILA LIFILLTAV VNMCTLMAM GFTIMAAIL FQRALIFIL WVTYGTCTT LAPHVGMGL RRDKRSVAL Premembrane WVTYGTCNQ YKCPHITEV 82 5 (Dengue 3) FKTASGINM 55 3 FKTASGINM 56 4 VLMMLVAPS LLAGFMAYM Premembrane LTPHSGMGL VFFVLMMLV (Dengue 4) LAGFMAYMI WVMYGTCTQ variants (envelope, capsid and premembrane) using a virtual matrix-based ProPred for peptide binding to 51 HLA-DR alleles. The analysis of these proteins was performed using the server ( raghava/propred/). The characterization of dengue virus variant antigens inducing CD4+ T cell epitopes response could critically contribute to the development of subunit vaccine. Computational analysis had been done by T cell epitope prediction software (TEPITOPE) to predict promiscuous HLA-DR ligand in structural protein 15. A study to evaluate the potential of synthetic peptides as possible flavivirus vaccine immunogens is underway. On the basis of computer analyses, synthetic peptides from the E glycoprotein of Murray valley encephalitis (MVE) and DEN 2 viruses were prepared, and their immunogenicity was evaluated in mice 16. T-cell analyses of synthetic peptides to other

5 SOMVANSHI & SETH: PREDICTION OF T CELL EPITOPES 197 viruses have correlated the association between T- and B-cell responses 17 the proliferative T-cell response or the antibody response to peptides 18,19. A new paradigm of vaccine design is now emerging, following essential discoveries in immunology and the development of bioinformatics tools for T cell epitope prediction from primary protein sequences. One rationale for this new paradigm is that following exposure to a pathogen, epitope specific memory T cell clones are established 20. Bioinformatics tools have the potential to accelerate research into the design of vaccines and diagnostic tests by exploiting genome sequences. In silico analysis could be combined with in vitro screening methods to speed up the identification of peptides that are immunogenic. Chemically synthesized domains of Foot and mouth disease virus (FMDV) VP1 were tested as potential peptide vaccine. The peptide corresponding to amino acids , and , which are located near the C- terminal end of VP1 and 9-24, and 25-41, and the N-terminal end of the VP1, were each bound to a separate insert carrier protein 21. It has been reported that several host cow, chicken, goose and human specific epitopes from the two significant surface proteins, hemagglutinin and neuraminidase, of Influenza A virus H5N1 for immunodiagnostic and vaccine development 22. The Plasmodium falciparum genome encodes more than 5300 proteins, each a potential target of protective immune responses. Immune responses to Plasmodium are dispersed over a large number of antigens an effective vaccine may have to include immunogenic epitopes of the genome, highly conserved across different strains and multi-stage life cycle. ProPred and ProPred1 were used to predict proteome promiscuous HTL and CTL epitopes 23. The prediction of surface proteins hemagglutinin and neuraminidase for the pathogenicity of birds to human for developing animal specific epitopes provides a suitable primary immunodiagnostic antigen for the detection of influenza A virus H5N1 24. The identification of T cells epitopes from the secretory and cell surface proteins of Mycobacterium tuberculosis H37Rv strain serves to define candidate antigens with vaccine potential. In 16 significant secretory cell surface proteins and virulent proteins, 69 promiscuous nanomer candidate epitopes for HTL were recognized against MHC Class II and 47 promiscuous epitopes against CTL for MHC class I 25. The 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 12. Future prospects for the availability of a vaccine that affords protection against all four serotypes offer the promise of an additional intervention to combat the disease in hyper endemic areas 26. The need for dengue vaccine that elicit sustained levels of neutralizing antibodies and provide long lasting protection in all vaccinated people without posing any risk to their health either by producing adverse effects or by inducing low levels of antibodies capable of enhancing subsequent dengue infections 27. Conclusion We have predicted several epitopes in the structural proteins of different dengue virus variants. They may be useful for the earlier diagnosis of dengue virus and dengue hemorrhagic fever. The conserved antigenic epitopes may be useful for the diagnosis of all the four variants of dengue and variants specific antigenic epitopes may be useful for specific diagnosis. The predicted epitopes may be used for safe subunit vaccine development against dengue fever and dengue hemorrhagic fever. The monitoring of dengue virus is required to prevent, control and manage dengue diseases. Acknowledgement We thank the Indian Council of Medical Research, New Delhi (64/2/07/BIF-BMS) and Department of Biotechnology, New Delhi for financial support. References 1 Rigau-Pérez J G, Gubler D J, Vorndam A V & Clark G G, Dengue surveillance United States MMWR CDC, Surveill Summ, 43 (1994) Gubler D J, Dengue and dengue hemorrhagic fever, Clin Microbiol Rev, 11 (1998) Henchal E & Putnak J R, The dengue viruses, Clin Microbiol Rev, 3 (1990) Nelson D R, Marousis C G, Ohno T, Davis G L & Lau J Y N, Intrahepatic hepatitis C virus-specific cytotoxic T lymphocyte activity and response to interferon alfa therapy in chronic hepatitis C, Hepatology, 28 (1998)

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