key: cord-0954304-zlwyiz0n
authors: Marchan, Jose
title: Conserved hla binding peptides from five non-structural proteins of sars-cov-2—an in silico glance
date: 2020-08-13
journal: Human Immunology
DOI: 10.1016/j.humimm.2020.08.001
sha: aa24f06d07df439766cb7eea5f3286bf90f45c4b
doc_id: 954304
cord_uid: zlwyiz0n

Abstract Coronavirus Disease 2019 (COVID-19) is a dangerous global threat that has no clinically approved treatment yet. Bioinformatics represent an outstanding approach to reveal key immunogenic regions in viral proteins. Here, five non-structural proteins (NSP) of SARS-CoV-2 (NSP7, NSP8, NSP9, NSP12, and NSP13) were screening for potential human leukocyte antigen (HLA) binding peptides. These peptides showed robust viral antigenicity, immunogenicity, and a marked interaction with HLA alleles. Interestingly, several peptides showed affinity by HLA class I (HLA-I) alleles that commonly activates to NK cells. Moreover, HLA-I and HLA class II (HLA-II) binding peptides induced humoral and cell-mediated responses after in silico vaccination. Notably, these peptides are conserved among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). These results may open further in vitro and in vivo investigations to develop novel therapeutic strategies against coronaviral infections.

1ab (ORF1ab), ORF3a, ORF6, ORF7a, ORF8, and ORF10. In turn, the ORF1ab expresses a 11 polyprotein whose proteolytical cleave forms 16 non-structural proteins (NSP, numbered 1 to 16) 12 [3] . The expression and activity of these NSP constitute a fundamental aspect for SARS-CoV-2 13 pathogenesis [2] . For instance, the protein complex formed by NSP7, NSP8, and NSP12 give rise 14 to the RNA polymerase, which is necessary for genome replication [4] , whereas NSP13 15 represents the virus helicase, which unwinds duplex RNA [4] . On the other hand, it is thought 16 that NSP9 binds to RNA, thereby improving viral replication [5] . 17 Several works have shown that NSP from other RNA viruses are key factors to elicit effective 18 immune responses [6, 7] . For instance, the presentation of conserved helicase peptides from 19 hepatitis C virus and certain flaviviruses in the context of human leukocyte antigen class I (HLA-20 I) is sufficient to inhibit viral replication [6] . Therefore, peptides derived from NSP represent a 21 valuable tool to develop future prophylactic and therapeutics approaches to fight viral diseases 22 such as COVID-19. However, our knowledge on the existence of high potential HLA binding 23 peptides from NSP of SARS-CoV-2 is still poor. Hence, this research aimed to identify 24 conserved and immunogenic HLA-I and HLA class II (HLA-II) binding peptides from five key 25 NSP of SARS-CoV-2, including NSP7, NSP8, NSP9, NSP12, and NSP13, using 26 immunoinformatics tools. 27 (www.ddg-pharmfac.net/vaxijen/) [11] , AlergenFP (http://ddg-51 pharmfac.net/AllergenFP/index.html) [12] , and ToxinPred 52 (http://crdd.osdd.net/raghava/toxinpred/) [13] , respectively. Moreover, HLA-II binding peptides 53 were also selected by their potential capability to induce interferon-gamma (IFN-g), which was 54 evaluated on IFNepitope web server (http://crdd.osdd.net/raghava/ifnepitope/) [14] . 55 

To observe how conserved were HLA binding peptides between SARS-CoV-2 and SARS-CoV 57 as well as between SARS-CoV-2 and MERS-CoV, an epitope conservancy analysis was 58 performed on the Immune Epitope Database and Analysis Resource (IEDB-AR) 59 (http://tools.immuneepitope.org/main/) [15] . Results were represented as a barplot using Rstudio The molecular docking simulation study was performed as follows. First, the three-dimensional 64 (3D) structures of 9-mer and 15-mer peptides were obtained from PEPFOLD server 65 (https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/) [16] . Second, the To evaluate the potential use of the HLA binding peptides on future vaccine trials, a vaccine 78 amino acid sequence was develop with the predicted HLA-I and HLA-II binding peptides, which 79 were linked using AAY and GPGPG linkers, respectively, as previously reported [22] . This pre 80 vaccine construct was subjected to immune response simulations on the C-ImmSim server 81 (http://150.146.2.1/C-IMMSIM/index.php) [23] . Three injections were applied four weeks apart 82 as described by Nain et al. 2020 [22] . The Simpson index D was used to interpret the diversity of 83 the immune response. (Table 1) . These 87 viral peptides showed robust viral antigenicity (≥0.5) and absence of either allergenic or toxic 88 residues ( Table 1 ). The higher number of peptides was observed in NSP12 and NSP13 (10 and 11 89 peptides, respectively). Interestingly, overlapping residues were observed among P7, P8, and P9, 90 and among P22, P23, P24, P25, and P26 (Table 1) . Regarding the HLA-I interacting alleles, most 91 of the viral peptides showed promiscuity by several HLA-I molecules including A*02:01, 92 B*08:01, C*02:02, and C*12:02 (Table 1) . 93 46 HLA-II binding peptides were predicted (numbered, P32 to P76). In this regard, 1 peptide was 94 identified for NSP7, 13 peptides for NSP8, 9 peptides for NSP9, 17 peptides for NSP12, and 6 95 peptides for NSP13 (Table 2 ). Similar to HLA-I binding peptides, strong viral antigenicity and 96 lack of allergenicity and toxicity were observed in these 15-mer viral peptides. Of note, each 97 HLA-II binding peptides was classified as a potential inductor of IFN-g (Table 2 ). Likewise, viral 98 peptides from NSP8, NSP9, NSP12, and NSP13 showed the presence of overlapping residues.

On the other hand, DRB1*01:01, DRB1*03:01, and DRB1*04:01were identified as the most 100 common HLA-II interacting alleles (Table 2) . showed 50% of conservancy (Fig 1) . In contrast, most of the SARS-CoV-2 peptides common to 104 MERS-CoV reached 50% of conservancy, with exception of two (P9 and P76) and four (P19, 105 P66, P67, and P68) peptides that showed 0% and 100% of conservancy, respectively (Fig. 1) . HLA binding patterns ( Fig. 2 and Fig. 3) .

Remarkably, these HLA-viral peptide complexes showed strong potential interactions (free 114 energy values < -6 kcal/mol -1 ) comparable to control peptides (Fig. 4A,B) . In this regard, P1, P5,   115   P10, P11, P12, P21, P25, P28, P29, P38, P39, P41, P42, P47, P52, P54, P55, P65, P68, P70, and   116 P76 showed the lower affinity values (Fig. 4A,B) . Interestingly, P52 obtained the lower free 117 energy value (-13.3 kcal/mol -1 ) compared to other HLA-II binding peptides (Fig. 4B) . 

Previous reports have been focus on uncover candidate targets for vaccine development from 131 structural proteins of SARS-CoV-2, including S, M, and E proteins [8, 24, 25] . However, the 132 presence of similar peptides from NSP7, NSP8, NSP9, NSP12, and NSP13 of SARS-CoV-2 had 133 remained poorly explored. The present study using an integrated in silico approach showed that 134 these five NSP harbour conserved viral peptides, whose properties-high viral antigenicity, 135 absence of allergenic and toxic residues, potential IFN-g induction, interaction with HLA alleles, 136 and suitable immune responses after in silico vaccination-make them potential targets for future The clear and proper interaction between conserved viral peptides and HLA alleles suggest that 142 they could elicit not only adaptive immune responses by CD8+ CTC and CD4+ THC but also an 143 innate immune response by Natural Killer (NK) cells-one of the first lines of defence against 144 viruses [27] . For instance, it has been shown a decisive role of activating killer immunoglobulin-145 like receptors (aKIR) in viral recognition by NK cells [28] . In this regard, Nayer and colleagues 146 reported that KIR2DS2 directly recognizes conserved viral peptides in the context of HLA-147 C*01:02, thereby leading to the inhibition of the hepatitis C virus and dengue virus replication 148 [6] . In the present study, several 9-mer viral peptides showed affinity by HLA-C*01:02 as well as 149 by HLA-C*02:02, which activates to KIRD2DS4 [28] . However, further research is required to 

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