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 Table of Contents  
ARTICLE
Year : 2016  |  Volume : 5  |  Issue : 1  |  Page : 34-43

Analysis of the DosR regulon genes to select cytotoxic T lymphocyte epitope specific vaccine candidates using a reverse vaccinology approach


1 DS Kothari Center for Research and Innovation in Science Education, Miranda House, University of Delhi; Department of Zoology, Miranda House, University of Delhi, Delhi, India
2 Open Source Drug Discovery Unit, Council of Scientific and Industrial Research, Delhi, India

Date of Web Publication8-Feb-2017

Correspondence Address:
Sadhna Sharma
Department of Zoology, Miranda House, University of Delhi, Delhi 110007
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.1016/j.ijmyco.2015.10.005

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  Abstract 


Objective/background: There is an urgent need for a more effective vaccine against Mycobacterium tuberculosis (Mtb). Although CD4+ T cells play a central role in host immunity to Mtb, recent evidence suggests a critical role of CD8+ T cells in combating Mtb. In the present study, we have predicted HLA antigen class I binding peptides of DosR operon using an in-silico approach. This method is useful as an initial computational filtration of probable epitopes based on their binding ability and antigenicity.
Methods: CD8+ epitopes were predicted by software NetMHC 3.4 and BIMAS. Self-peptides were found and excluded by indigenously developed Perl script. Antigenicity of promiscuous peptides was predicted using a VaxiJen server. The top VaxiJen scoring antigenic peptides were docked to globally relevant HLA allele using CABS dock and Hex program.
Results: A total of 1436 overlapping nonamer peptides were generated which gave 46 promiscuous epitopes, 25 were predicted to be antigenic. Rv2627 epitope “SAFRPPLV” which gave the highest Vaxijen score of 1.9157 and showed binding to all the three HLA loci. The top VaxiJen scoring antigenic peptides were docked and had significant interactions with residues of the HLA class I molecule indicating them to be good cytotoxic T lymphocyte epitopes.
Conclusion: Our study has generated several promiscuous antigenic peptides capable of binding to major histocompatibility complex class I with high affinity. These epitopes can become part of a postexposure multivalent subunit vaccine upon experimental validation.

Keywords: Cellular immunity, CD8+ T cell epitopes, DosR operon, Mycobacterium tuberculosis, TB vaccines


How to cite this article:
Pandey K, Sharma M, Saarav I, Singh S, Dutta P, Bhardwaj A, Sharma S. Analysis of the DosR regulon genes to select cytotoxic T lymphocyte epitope specific vaccine candidates using a reverse vaccinology approach. Int J Mycobacteriol 2016;5:34-43

How to cite this URL:
Pandey K, Sharma M, Saarav I, Singh S, Dutta P, Bhardwaj A, Sharma S. Analysis of the DosR regulon genes to select cytotoxic T lymphocyte epitope specific vaccine candidates using a reverse vaccinology approach. Int J Mycobacteriol [serial online] 2016 [cited 2021 Jun 18];5:34-43. Available from: https://www.ijmyco.org/text.asp?2016/5/1/34/199740




  Introduction Top


Tuberculosis (TB) is a major infectious disease of global concern, which has afflicted humans throughout the recorded history [1],[2]. The World Health Organization ranks TB among the top 10 causes of human mortality in the world.

To protect against TB, vaccination with the attenuated Mycobacterium bovis Bacillus Calmette–Guérin (BCG) has been practiced worldwide for several decades. However, BCG vaccination has shown highly variable efficacy to protect against the most infectious form of disease in adults, that is, pulmonary TB [3],[4]. In addition, the diagnostic value of the present skin test reagent purified protein derivative of Mycobacterium tuberculosis (Mtb) is not very reliable because of antigens that are cross-reactive with environmental mycobacteria and the vaccine strains of M. bovis present in BCG. Furthermore, BCG being a live attenuated vaccine can become virulent and cause disease in immune compromised individuals, such as AIDS patients [3]. Hence, identification of antigens that are useful for the specific diagnosis and development of improved vaccines with respect to greater safety and protective efficacy globally is an urgent need [4].

Mtb is an intracellular pathogen, against which a cell mediated immune response whether a CD4 or CD8 is mounted. Response of a T-cell is dependent upon the processing of the antigen into 8–15 amino acid long peptides. These peptides then interact with the major histocompatibility complex (MHC). Different peptides bind to the MHC molecule with different affinity and off-rate (the duration of peptide binding to the MHC molecule) [5]. The last step is recognition of this tri-molecular complex of MHC–β2 microglobulin–peptide by T cells. Two factors play a decisive role when an antigen is presented by MHC molecules: (1) the nature of the presenting MHC allele; and (2) the amino acid (AA) composition of the target peptide.

In the past, the focus of vaccine development has been on early phase proteins like Ag85, ESAT-6, CFP-10, and so forth, expressed during the initial phase of Mtb infection. However, Mtb late phase proteins like Dormancy Survival Regulon (DosR) proteins that are expressed under intracellular adverse conditions have also been shown to induce a strong T cell and interferon-γ response and, therefore, could prove to be good vaccine candidates [3],[6],[7],[8],[9],[10],[11]. In our previous study, several DosR regulon latency antigens were shortlisted as good vaccine candidates [3]. Some of these proteins show a good CD8 cell response in our laboratory (unpublished data), wherein Rv2627 elicited highest response. In the present study, we wish to bioinformatically identify promiscuous antigenic CD8+ cell epitopes derived from the six selected DosR proteins. The identified epitopes may then be included in an epitope-bound vaccine design.

Although mainly CD4+ Th1 cells elicit a protective immune response against Mtb, experimental evidence also suggests a role for CD8+ T cells in both active and latent TB [12],[13]. Mice studies involving adoptive transfer of CD8+ T or their in vivo depletion have shown that this subset could confer protection against subsequent challenge of Mtb [14]. Mice deficient in components of MHC class I were also found to be more susceptible to the infection [15]. Caccamo et al [13] have shown that healthy people with latent TB infection have a high frequency of Mtb antigen specific CD8+ T cells, thus, showing a protective and long lived immune response [13]. Therefore, it is worth investigating important CD8+ T cell epitopes that help mount a cellular immune response against infected macrophages.


  Materials and methods Top


Prediction of physiochemical properties

The complete AA sequences of the six proteins Rv2029, Rv2031, Rv2032, Rv2626, Rv2627, and Rv2628 of Mtb were obtained from the TubercuList database [16]. The six proteins were analyzed for their subcellular localization with the PsortB software [17]. The transmembrane region was predicted using TMHMM and HMMTOP [18],[19]. The presence of a signal sequence was predicted using the SignalP software [20].

Prediction of CD8 epitopes

The selected DosR protein sequences were passed through two software servers, BIMAS and NetMHC 3.4, which generate overlapping epitopes by a step size of one residue. The length of the epitopes was selected to be nine AA MHC class I preferentially bind nonamers.

BIMAS uses HLA BIND prediction algorithm and estimates the binding against nine HLA-A alleles, 20 HLA-B alleles, and four HLA-C alleles. For BIMAS a T1/2 of 100 min was chosen as a cut-off point in order to select the relatively high-affinity binding peptides [21],[22].

NetMHC Server predicts epitopes for 34 alleles of HLA-A, 33 alleles of HLA-B, and 10 alleles of HLA-C using artificial neural networks [23],[24],[25]. Only strong binders with half maximal inhibitory concentration less than 500 (≤IC500) were selected from the total epitopes generated.

Identification of “self” peptides

Each of the 9-mer peptides was analyzed for similarity with any of the human proteins annotated so far. An indigenously developed Perl script was applied to identify self-peptides that exhibited: (1) 100% (9AA); (2) 90% (8AA); and (3) 70% (7AA) similarity with the nonamer peptides of the human proteome. The self-antigens were excluded from further analysis.

Prediction of promiscuous epitopes and their antigenicity

Promiscuity means a given MHC molecule can bind numerous different peptides, and a peptide motif is recognized by different MHC molecules. For BIMAS, epitopes that bound to three or more alleles, and bound to five or more HLA alleles for NetMHC, were selected as promiscuous epitopes. Vaxijen server was used to predict the antigenicity of these promiscuous peptides [26].The peptides that gave a score of 0.4 or above were shortlisted and two top scorers for each protein were used for docking studies.

Peptide MHC docking by CABS dock and Hex

The binding of top scoring antigenic promiscuous peptides to their respective class I alleles was investigated by docking. Two MHC Class I alleles, A2 supertype (PDB-1S9Y) and B_5101 (PDB-1E27) were used to carry out docking because of their global presence. Firstly, the docking was carried out by the CABS dock server [27] and the linear peptide and MHC molecule were separated from the docked structure. The linear peptide and MHC molecule were than docked again using Hex to obtain an energy score [28]. The control peptide was a cytotoxic T lymphocyte CTL epitope (SLLMWITQS) belonging to NY-ESO-1 testicular cancer antigen for A2 supertype allele. For B_5101 allele, it was an immuno-dominant epitope (LPPVVAKEI) of human immunodeficiency virus. Different peptide-MHC interactions were visualized using Discovery Studio Visualizer version 4.1 (Accelerys Inc., San Diego, California, USA).


  Results Top


Physiological characterization of proteins

Using PsortB, it was found that three proteins (Rv2627, Rv2628, and Rv2032) belonged to cytoplasm, whereas Rv2029 had an association with cytoplasmic membrane. Rv2031 was localized in the cell wall and location of Rv2626 could not be predicted. By using the TMHMM and HMMTOP software it was found that the proteins Rv2627 and Rv2029 possess two and one transmembrane helices respectively, oriented to cytoplasmic side. None of the six proteins possessed a signal peptide sequence.

Total number of epitopes generated by BIMAS and NetMHC

A total of 1,436 overlapping nonamer peptides were generated from six DosR proteins with a step size of one residue. Using BIMAS, a total of 205 epitopes binding to 33 alleles were selected with the described cutoff. Rv2627 gave the highest number of MHC binding epitopes (62), followed by Rv2032 (42), and then Rv2029 (39).

NetMHC generated a total of 332 strong binding epitopes for 78 alleles. Rv2627, again, gave the highest number of epitopes (95) binding to MHC class I, followed by Rv2032 (83) and Rv2029 (48).

Self-antigen determination

One self-peptide from Rv2031 “GRYEVRAEL” and Rv2032 “EALLGCGAV”, and 24 peptides with 7 AA similarity were removed from further analysis.

MHC class I binding profile of the DosR proteins

Specificity and promiscuity of peptide MHC class I allele binding

In BIMAS, 25 peptides bound to three alleles and only one epitope (“HPREQVTRL”) of Rv2627 bound to five alleles from six DosR proteins. In NetMHC, 20 epitopes bound to five or more alleles. The epitope “YIASLVASL” of Rv2627 bound to a maximum of 13 alleles ([Table 1] and [Table 2]).
Table 1: Predicted peptide major histocompatibility complex binding profile of select DosR genes using BIMAS.

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Table 2: Predicted peptide major histocompatibility complex binding profile of select DosR genes using NetMHC.

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Epitope density of proteins

Epitope density is the ratio of number of MHC binding peptides of a protein to the total number of overlapping nonamers generated from that protein. Rv2628 was found to have the highest epitope density and Rv2029 the lowest epitope density in the software ([Table 1] and [Table 2]).

Proteins which bind to a large number of alleles

Rv2029 bound to a maximum of 18 alleles in BIMAS and 45 alleles in NetMHC, whereas Rv2628 bound to the lowest (10) alleles in BIMAS and 29 alleles in NetMHC ([Table 1] and [Table 2]).

Alleles binding to maximum number of epitopes

In BIMAS, the allele B_2705 bound to maximum number of nonameric peptides of the select DosR proteins followed by B_5102, B_5101, and B_5103. The alleles A_1101, A_3101, A_3302, B_3902, Cw_0602, and Cw_0702 did not bind to any of the peptides ([Figure S1]).

In NetMHC, allele A_0250, A_0211, A_0216, B_1503, B_2720, B_3501, C_1203, and C_1402 showed maximum binding to the epitopes. The alleles A_6601, B_0803, and B_4601 did not bind to any epitope ([Figure S2]).

Prediction of antigenicity of promiscuous epitopes

Only 25 of the 46 promiscuous epitopes from the six DosR proteins gave a good Vaxijen score. Promiscuous epitope “SAFRPPLV”Rv2627 predicted by both the software gave the highest Vaxijen score of 1.9157. Interestingly this predicted antigenic epitope showed binding to all the three HLA loci, that is, A, B, and C ([Table 3] and [Table 4]).
Table 3: Binding profile of two top VaxiJen scorer promiscuous antigens in BIMAS.

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Table 4: Binding Profile of Two Top VaxiJen Scorer Promiscuous Antigens In NetMHC.

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Docking of promiscuous epitopes to globally relevant alleles

Seven epitopes for A2 supertype and seven epitopes for B_5101were docked and their energy scores were found comparable to that of the control peptide. For A2 supertype, epitope “VTFGRDFPV” gave the maximum energy. The anchor residues at position second and ninth were found to have significant hydrophobic interactions with MHC molecule. Other relevant interactions are also depicted in [Figure 1]A and [Table 5] and [Table 6].
Table 5: Docking of promiscuous antigenic peptides with A2 supertype (1S9Y).

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Table 6: Docking of promiscuous antigenic peptides with B_5101 (1E27).

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Figure 1: Two different class I MHC molecules with nonameric peptides and their anchor residues (Green) Anchor residues, at positions 2 and 9, that interact with the class IMHC molecule tend to be hydrophobic amino acids. The two MHC proteins bind peptides with different anchor residues. Contact with theMHCmolecule is by hydrogen bonds to anchor residues 2 and 9. Note. Ala = alanine; Arg = arginine; Asp = aspartic acid; Cys = cysteine; Gly = glycine; His = histidine; Ile = isoleucine; Leu = leucine; Lys = lysine; Met = methionine; MHC = major histocompatibility complex; Phe = phenylalanine; Pro = proline; Ser = serine; Trp = tryptophan; Tyr = tyrosine; Val = valine.

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For allele B_5101, the epitope “DAMVAAITV” of Rv2029 gave maximum binding energy which can be attributed to the four hydrogen bonds formed by the peptide at position Asp1, Met3, Ala5, and Thr8, with Asn77, Asn70, Tyr99, Glu55, and Val34 of the MHC respectively ([Figure 1]B, [Table 3] and [Table 4]). The epitopes have hydrophobic AA at the second and ninth position as these residues help in anchoring the peptide into the MHC groove as shown in [Figure 2]A and B.
Figure 2: Docking of peptides with globally relevant alleles. A) Ball and Stick model of epitope VTFGRDFPV (yellow) docked onto the A2 Supertype (1S9Y). The inset shows the detailed interactions of the epitope (pink) with the MHC residues (blue).Potential bonds formed between the two are shown as broken lines (Green- Hydrogen, Orange- Electrostatic and Purple-Hydrophobic). Second residue is Threonine and last residue is Valine. B) Ball and Stick model of epitope DAMVAAITV (yellow)docked onto the B_5101 (1E27). The inset shows the detailed interactions of the epitope (pink) with the MHC residues (red).Potential bonds formed between the two are shown as broken lines (Green- Hydrogen, Orange- Electrostatic and Purple-Hydrophobic). Second residue is Alanine and last residue is Valine.

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  Discussion Top


There is increasing experimental evidence to support that CD8+ T cells provide immunity by releasing the proinflammatory cytokines and lysing Mtb infected cells. To activate CD8+ T cells, mycobacterial antigens need to enter the class I MHC pathway before they can be processed and presented by class I MHC on the cell surface. In spite of having support for the role of CD8+ cells in protective immunity, very few CTL epitopes of Mtb are known. It was shown by Andersen [29] that there is a huge expansion of CD8+ T cells in the later stages of infection and this could have implications for the development of postexposure vaccines against latent TB [29].

Recently, some TB vaccines are being made by incorporating the late phase DosR regulon latency antigens as a strategy to improve the efficacy of BCG. ID93 and H56-IC31 are two vaccines currently under different stages of trial which have incorporated DosR antigens, Rv1813c and Rv2660, respectively [3]. The DosR regulon has some 50 genes under it and examining the antigenic potential of each protein experimentally is an expensive and tedious process. Therefore, an initial computational filtration of probable CTL candidate antigens would be cost and time effective. Reverse immunology based on bioinformatics tools to predict relevant epitopes, followed by in vitro/ex vivo validation of selected epitopes would be a better solution.

Humans possess three HLA gene loci A, B, and C, which are highly polymorphic and are codominantly expressed [11]. MHC class I is restricted by epitopes which are 8–10 AA long. Nonamers have higher binding affinity for MHC class I in comparison to longer or shorter peptides [11]. Thus, in our study the length of the peptide was kept to nine AAs. Most of our MHC class I epitopes had hydrophobic AAs at the second and ninth position as these residues help in anchoring the peptide into the MHC groove. The second, third, fifth, and ninth residues of the peptides interacted with the MHC molecules, whereas the remaining residues may interact with T-cell receptors which corroborates with the previous findings [11].

None of the six proteins were found to be secretory in nature; two proteins, Rv2627 and Rv2029, had transmembrane domains but were located on the cytoplasmic side, whereas the remaining four proteins were located in the cell cytoplasm. Previously, only secretory proteins were considered to be antigenic; however, nonsecretory or somatic proteins can be processed by MHC class I machinery and presented to CD8+ T cells [30].

NetMHC server distinguishes between strong and weak binding epitopes. There is a clear bias towards HLA-A binders in this server with 413 strong binding epitopes, whereas HLA-B or HLA-C allele bound to 245 and 87 epitopes, respectively. For BIMAS, the affinity with which a peptide bound to an allele was in the range of 100–6000 minutes and 53 epitopes had T1/2 of 500 min or more. In our study, we found that BIMAS gave a higher number of epitopes binding to B allele and that was with stronger binding affinity in comparison to other loci. It has been reported that HLA-B-restricted epitopes elicit stronger CD8+ T-cell response in Mtb infection, as well as in many viral infections [5],[14],[31],[32],[6].

The self-peptides found in our DosR proteins shared homology with polycystin-1 isoform X8 and mitogen-activated protein kinase kinase kinase 9 isoform X3 respectively. These self-peptides are poor binders to their cognate class I molecule as the number of T cells that might recognize them will be diminished due to their thymic and peripheral deletion [33]. They might also mount an autoimmune response on recognition [11],[15].

It was observed that BIMAS had 17 alleles, whereas NetMHC had 33 alleles which are predominant in North Indian populations. Five alleles in HLA-A, five in HLA-B, and one in HLA-C were found to be overlapping in both the software. The alleles which bound to a large number of epitopes were A_0211, A_6801, A_0206, C_1203, A_3101, and B_3501 and their frequency coverage ranged from 3.3% to 22.2% in North India. This information is important as India shares 24% of global TB burden according to a World Health Organization report [34].

For a good vaccine design, promiscuous epitopes are of greater significance as they have broad specificity in terms of HLA binding. Only 12.8% in BIMAS and 6.3% in NetMHC were promiscuous epitopes out of which 54% were antigenic in nature, making them important in the vaccine development process. Fourteen top scoring antigenic epitopes were docked with either HLA-A2 allele, having a global frequency of 80% and almost 40% frequency in North India, or B_5101 allele, which strongly bound to majority of the epitopes. [6],[35],[36],[37],[38],[39]. The energy score of the docked structures was not only comparable to the cocrystallized control epitopes of the two referred alleles but also to the previously reported Mtb secretory epitopes, “YMLDMTFPV” of Rv3036 and “LGKAMTNLL” of Rv3812, which bound to alleles A_0201 and B_5101, respectively.

MHC class I alleles have six pockets from A–F, each of which has residues which are either conserved or polymorphic and play an essential role in peptide MHC interactions [33]. It is reported that specificity for class I binding is largely conferred by two or three dominant anchor residues, while antigen specificity of MHC-peptide complex recognition is generally determined by the few side chains of the peptide antigen that are solvent-exposed (between one and three residues) and available for T-cell receptor contacts [40]. All our docked epitopes of A2 allele interacted with the different residues found in the six pockets. Residues at second and ninth positions of the epitopes are hydrophobic which facilitates the peptide's interaction with the pocket B and F of the MHC allele, respectively. As reported in literature, all our epitopes binding to B_5101 were also found to have alanine at the second position and valine or isoleucine at the ninth position.

It is the hydrogen-bonding which secures the peptide's N-terminus and C-terminus independent of peptide sequence. It also serves as a bridge between the peptide and buried hydrophilic MHC residues [41]. Hydrophobic interactions between MHC and peptide are the driving force for the specific binding, whereas charged residues form strong electrostatic interactions which are essential in strengthening the peptide MHC interactions [41]. We observed that the interactions between the epitope and the allele were similar in both CABS dock server and Hex programs. This further substantiates the candidature of selected epitopes for incorporation in future vaccines.

We have tested the immunogenicity of these six proteins and found it was Rv2627 which elicited maximum CD8 response (unpublished data). This bioinformatics study supports the same and provides us with promiscuous and antigenic epitopes of not just Rv2627 but the other five proteins which are expressed during latent TB capable of binding to MHC class I with high affinity. These epitopes can become part of a postexposure multivalent subunit vaccine upon experimental validation. The antigenicity of these peptides is currently being tested experimentally in our laboratory using peptide microarray and MHC tetramer technology.

Conflicts of interest

The authors have no competing interests to declare.

Acknowledgments

We would like to acknowledge Dr. Pratibha Jolly, Principal, Miranda House and PI, DS Kothari Centre for Research and Innovation in Science Education, Miranda House, University of Delhi for providing infrastructural support. Also, the financial support of the Department of Biotechnology, Government of India (Grant No. BT/PR5638/MED/29/580/2012) and ICMR for Senior Research Fellowships to IS and SwS is greatly acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmyco. 2015.10.005.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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