key: cord-0986009-oqtu87f5 authors: Pritam, Manisha; Singh, Garima; Swaroop, Suchit; Singh, Akhilesh Kumar; Pandey, Brijesh; Singh, Satarudra Prakash title: A cutting-edge immunoinformatics approach for design of multi-epitope oral vaccine against dreadful human malaria date: 2020-04-29 journal: Int J Biol Macromol DOI: 10.1016/j.ijbiomac.2020.04.191 sha: 28db8e9fcf106e7e3392f627f6f5770e180ec33a doc_id: 986009 cord_uid: oqtu87f5 Abstract Human malaria is a pathogenic disease mainly caused by Plasmodium falciparum, which was responsible for about 405,000 deaths globally in the year 2018. To date, several vaccine candidates have been evaluated for prevention, which failed to produce optimal output at various preclinical/clinical stages. This study is based on designing of polypeptide vaccines (PVs) against human malaria that cover almost all stages of life cycle of Plasmodium and for the same 5 genome derived predicted antigenic proteins (GDPAP) have been used. For the development of a multi-immune inducer, 15 PVs were initially designed using T cell epitope ensemble which covered >99% human population as well as linear B cell epitopes with or without adjuvants. The immune simulation of PVs showed higher levels of T-cell and B-cell activities compared to positive and negative vaccine controls. Furthermore, in silico cloning of PVs and codon optimization followed by enhanced expression within Lactococcus lactis host system was also explored. Although, the study has sound theoretical and in silico findings, the in vitro/in vivo evaluation seems imperative to warrant the immunogenicity and safety of PVs towards management of P. falciparum infection in the future. CamSol and SOLPro. The analysis of secondary structure elements (alpha helix, extended strand and random coil) were performed using PSIPRED tool. Further, tertiary structure analysis was carried using tools ModRefiner and PROCHECK. The biological function and allergenicity were evaluated based on tools DeepGOPlus and AllergenFP, correspondingly. The best docked complex (in terms of lowest docking energy) PVs with receptors TLR2 and TLR4 were chosen for immune simulation study using C-ImmSim tool along with two positive vaccine controls (C3, C4) as mentioned in section 2.6 and one negative vaccine control (C5) so as to compare the simulation results. The C5 was designed using suitable linkers as well as non-binding HLA class I and II epitopes by applying the same strategies as used in PVs. The non-epitopes were The molecular docking between antibodies IgG1 (PDB ID: 6B5L) as well as IgG3 (PDB ID: 5BK0) with PVs (PV1A/PV3B) were performed using ClusPro 2.0 tool along with co-crystallized respective control epitopes NPDPNANPNVD (C6, IEDB ID: 756359) and NANPNANPNANPNANPNANP (C7, IEDB ID: 43248) of Pf CSP [56, 57] . Molecular dynamics of top 2 docked complexes PV1A-TLR2 and PV3B-TLR4 were performed through iMODS server to explain the collective protein motion in the internal coordinates through normal mode analysis (NMA). The NMA in dihedral coordinates naturally mimics the combined functional motions of protein molecules modelled as a set of atoms connected by harmonic springs [58] . J o u r n a l P r e -p r o o f 9 The DNA coding sequences of the oral PVs (PV1A and PV3B) were optimized for elevated protein expression using Java Codon Adaptation Tool (JCat) following options were selected: i) Lactococcus lactis (strain IL1403) as expression host, ii) avoid rho-independent transcription terminators, iii) avoid prokaryotic ribosome binding sites and iv) avoid cleavage sites of restriction enzymes. Further, for in silico cloning of PV1A and PV3B cDNA (with stop codon) SnapGene software was used involving insertion at restriction site of FspI (6006) in plasmid vector pIL1 (Gene bank accession number: HM021326) [59] . According to VIOLIN database (accessed on June 26, 2019), total 16 vaccines available so far for against P. falciparum from different life-cycle stages, but they have not succeed to get approval from FDA, USA for world-wide marketing [60] . The RTS,S/AS01 is the only world's first European Medicines Agency (EMA) approved malaria vaccine with partial protection in young children (36.3%) for use to only Sub-Saharan African region along with severe adverse effect (24.2%-28.4%) and incurable adverse effect (1.5%-2.5%) [61, 62] . In addition, the efficacy was further declined to almost zero after 4 th year and negative in 5 th year [63] . The aforementioned facts warrant exhaustive efforts/research towards the development of a more effective PV that can elicit robust immune response globally. The present study is an extension of our previous report [24] that exploits 5 homologous antigens conserved among human malaria parasites P. falciparum, P. vivax, P. ovale and P. malariae (with minimum 38.62% identity recognized through BLASTp tool) as potential platform for designing of PVs [64] . In recent years, epitope based designing of vaccine is a new strategy that has been employed by world-wide researchers towards the development of efficient PVs against numerous diseases such as leishmaniasis, malaria and so on. In this context, the exploitation of computational approaches is not only cost-effective for vaccine development but also diminishes time period and risk of failure in experimental studies [26, 27, 65, 66] . In this study, 82 continuous B cell epitopes were forecasted from 5 GDPAP using BCPREDS (Supplementary Table S1 ). These 82 continuous B cell epitopes were found to possess total 433 T-cell epitopes including 142 HLA class I epitopes and 291 HLA class II epitopes (Supplementary Table S2 ). These T-cell epitopes were forecasted from the pool of predicted continuous B-cell epitopes as the antigen presentation to T-cells was supposed to be more J o u r n a l P r e -p r o o f efficient if it is recognized by the B-cell. In addition, an antigen-specific B-cell may present multiple T-cell epitopes to the immune system and, thus enhances its ability to be triggered in a specific manner [67] [68] [69] . Further, based on the PPC analysis an epitope ensemble of 13 HLA class I epitopes with 98.75% and 3 HLA class II epitopes with 56.85% world coverage were designed using criteria described previously (Table 2 ) [24] . However, a combined set of 16 were also reported in similar study conducted by Pritam et al. [24] . In case of malaria, adaptive immune system elicits both cellular and humoral immune responses, which are associated with B and T lymphocytes, respectively. However, mainly the CD4+ T lymphocytes (also known as helper T cell (Th), Th1 and Th2) elicit IFN-γ and IL-4, correspondingly) regulate the malaria infection [68, 70] . Besides these, TLRs are also involved in the activation of different signalling cascade that ultimately express the genes of pro-inflammatory cytokines like IFN-γ, etc. [71] . The IFN-γ is associated with depletion of liver-stage parasites [72, 73] . This is also supported by present study, where the epitopes T2, T7, T8, T10, T11 and T1, T2, T3, T4, T5, T6, T7, T8, T9, T11, T12, T13, T14, T16 were found to induce the IFN-γ and IL-4 responses, correspondingly (Supplementary Table S3 ). Amongst aforementioned epitopes ensemble, the T14 was recorded as one of the potent candidate to induce IL-10 response that found to suppresses the pathogenic inflammatory responses concerning control of malaria parasite [74] . Linear B-cell epitopes is linked to antibody generation, where identification of such epitopes using traditional approaches is not only costly but also time consuming with involvement of difficult processes [75] . In order to overcome aforementioned issues, the present study involved the prediction of T-cell epitopes using linear B-cell epitopes as input instead of whole antigen so as to minimize not only the size of PV but also elicit both cellular (T cell epitope) as well as humoral (Bcell epitope) immune responses. Further, the non toxic nature of adjuvants A and B also helps in production of several cytokines (e.g., INF-γ, TNF-α, IL-2, IL-4, IL-6, IL-12) through induction of dendritic cell, B cell, macrophage and T-cell, which ultimately boost the concentration of the antibodies reported in several studies linked to various disease causing agents including human rotavirus, HIV, Helicobacter pylori, Influenza virus [76] [77] [78] [79] . Therefore, 15 PVs were designed through epitope ensemble of T cell epitopes and/or linear B-cell epitopes having epitope ensemble with different linkers as well as adjuvants, which are responsible for the activation of TLR2 and TLR4 receptors pertaining to malaria. Initially, five non-adjuvant PVs (PV1-PV5) were designed followed by incorporation of TLR2 and TLR4 binding specific adjuvants that resulted into respective design of 10 adjuvant PVs, i.e., PV1A-PV5A and PV1B-PV5B (Table 3) . Further, EAAAK linker was incorporated at N-terminal of PVs as it is stiff and prevents the assembly of adjuvant with other vaccine domain [80, 81] . Although, the adjuvants are found to enhance the immunogenicity of vaccines but they may cause toxicity/adverse reaction. Therefore, we have designed 5 PVs without adjuvants, where the designing of PV1 having only T cell epitopes (HLA J o u r n a l P r e -p r o o f class I and II) and they were joined together by using linker L 2 and L 3 . Likewise, in PV2, we have exploited merely linear B-cell epitopes attached together with linker L 3 . Similarly in PV3, both Tand B-cell epitopes were joined with linkers L 2 and L 3 while, in PV4, we have exploited merely linear B-cell epitopes attached together with linker L 2 . Amongst these two linkers, L 3 is a universal linker, which can enhance the proteasome processing along with immunogenicity, while L 2 is a flexible linker that can stimulate better immune response [42, 77, 82] . As exemplary vaccine is found to induce multi-immune response (B-and T-cell immune response), therefore in the designing of further PVs both the T-and B-cell epitopes were used so as to elicit humoral/cellular response [83] . The PV3 and PV5 were differing from each other with respect to linkers L 3 and L 2 , respectively used for joining continuous B-cell epitopes. However, in case of designing a negative polypeptide vaccine control, linkers L2 and L3 were employed to connect non-HLA class I and II T cell epitopes (Table 3 ). -T2-L2-T5-L2-T6-L2-T9-L2-T11-L2-T12-L2-T13-L2-T1-L2-T4-L2-T8-L2-T10-L2-T4-L2-T3-L2-T7-L3-T14-L3-T15-L3-T16-L3 4 PV2 299 T2-L2-T5-L2-T6-L2-T9-L2-T11-L2-T12-L2-T13-L2-T1-L2-T4-L2-T8-L2-T10-L2-T4-L2-T3-L2-T7-L3-T14-L3-T15-L3-T16 14 PV5A 643 L1-A-L1-T2-L2-T5-L2-T6-L2-T9-L2-T11-L2-T12-L2-T13-L2-T1-L2-T4-L2-T8-L2-T10-L2-T4-L2-T3-L2-T7-L3-T14-L3-T15-L3-T16-L2-B2-L2-B5-L2-B6-L2-B8-L2-B9-L2-B10-L2-B9-L2-B1-L2-B4-L2-B3-L2-B7-L2-B11-L2-B5-L2-B13-L2 15 PV5B 649 L1-B-L1-T2-L2-T5-L2-T6-L2-T9-L2-T11-L2-T12-L2-T13-L2-T1-L2-T4-L2-T8-L2-T10-L2-T4-L2-T3-L2-T7-L3-T14-L3-T15 A and B) , T-cell epitopes (T1-T16), and B-cell epitopes (B1-B13). The TLRs, especially the surface one, viz. TLR2 as well as TLR4 are available not only on the immune cells, but also on epithelial cells and fibroblasts that recognizes PAMPs and bridge the innate as well as adaptive immunity of the host by regulating the balance between Th1 and Th2 (50s ribosomal L7/L12) in designed PVs could be the good choice against P. falciparum [77, 78, 86, 93] . Even combining two distinct TLR agonists into an adjuvanted subunit vaccine have showed synergetic protective efficacy [94, 95] . Altogether, these facts led to the hypothesis of using both TLR2 and 4 receptors agonists A and B, respectively in the designed PVs and subsequently docking experiment was performed to reveal the possible association among PVs and TLR [96, 97] . For molecular docking, the tertiary structures of 15 PVs were predicted that revealed more than 80 % of amino acids in favoured regions. Overall 22 docking studies were carried out using ClusPro2.0 tool including control C1 and C2 against receptors TLR2 and TLR4, respectively (Table 4 ). This resulted into total 18 docked models, i.e., M1 to M18 including 16 PVs and 2 controls. It is quite interesting to note that the PVs designed without adjuvants were also able to interact (dock) with TLR2 and TLR4 (having good energy scores) over control except PV3. Therefore, they might be capable to elicit innate immunity [98] [99] [100] , which are in well agreement with earlier studies regarding the rapid production of IFN-γ [101, 102] . Amongst 15 designed PVs, PV3, PV5A and PV4B were not able to dock by ClusPro tool with their respective receptors. So, a total 12 potential PVs with 16 docked models were obtained for TLR2-TLR1 (M2-M9) and TLR4-MD2 (M11-M18). The docking energy of control models M1 (-685.9 Kcal/mol) and M10 (-794.9 Kcal/mol) for complexes TLR2-TLR1-C1 and TLR4-MD2-C2 were found higher over designed potential PVs, which indicates that all the docked PVs have formed stronger immunological complexes over control ligands. Amongst the designed PVs without adjuvants (PV1, PV2, PV4 and PV5), PV4 showed the lowest docking energies -1047.4 Kcal/mol and -1070.7 Kcal/mol with respect to TLR2 and TLR4 receptors, correspondingly. These clearly indicated that the PVs without adjuvants have interacting domain to induce innate immune system. This is in agreement with the recent study where human TLR4-derived self-assembling peptide nanoparticles have been used as non toxic vaccine adjuvant with filarial antigenic protein to induce the immunological responses in mice [103] . Besides these, the linker L2 has been utilized in the designing of PV1A, PV3B and PV4 (Table 4 ). However, based on overall docking J o u r n a l P r e -p r o o f 18 score, PV1A (-1275.5) and PV3B (-1269.2) against receptors TLR2 and TLR4, respectively were selected as leading PVs for further structural and functional analysis (Fig. 4) . were forecasted by AllergenFP tool at threshold value > 0.8. Also, the secondary structure analysis (SSA) of a protein is beneficial for understanding its folding, stability as well as function [106] [107] [108] [109] [110] . In this context, the present study revealed alpha helices Table S4 ). These leading PVs were also predicted to be involved in multi-organism process as well as cell adhesion and immune system process, respectively, as predicted by DeepGOPlus tool which is based on deep convolutional neural network model and Gene Ontology (GO) scheme. The overall structural and functional analysis of leading PVs showed comparatively similar properties over positive vaccine controls C3 and C4 (Table 5) . Thus, the leading polypeptide vaccines PV1A and PV3B have the capability to J o u r n a l P r e -p r o o f 20 induce both humoral as well as cellular immune responses. However, the orally administered polypeptide vaccines suffer from the poor stability, insolubility, weak bioavailability and low immunogenicity due to acidic environment of the upper GI-tract and inefficient delivery to the mucosa-associated lymphoid tissue. Therefore, genetically engineered L. lactis expression host can be used for production and delivery of vaccine antigens due to several advantageous properties viz. easy and safe production as well as storage, survival in gastric environment and self-adjuvanticity [112, 113] . Multiorganism process Immune system process and cell adhesion In the course of human malaria infection, pro-inflammatory (TNF-α, IFN-γ and IL-12) and antiinflammatory (IL-4 and IL-10) cytokines were produced by Th1 and Th2 cells, respectively [114] . In addition, cytotoxic T lymphocyte, natural killer cells and macrophages were activated by elicitation of IL-4, which helps to control pathogen effect [115, 116] . Even, the most successful vaccine candidate of malaria, RTS,S was reported to elicit IFN-γ, IL-2, IgG titers, activation of CD4+ T cell responses [72, 117] . In this background, the present study involved the immune simulations of PV1A and PV3B using C-ImmSim tool along with the positive vaccine controls (C3, C4) ( Table 6 ). The C3 is a self-assembling polypeptide nanoparticle (SAPN) based P. falciparum When an antigen interacts with antibody it induces the humoral immune response and helps in clearance of pathogen. The IgG antibodies (named in order of decreasing abundance IgG1, IgG2, IgG3, and IgG4) are one of the most abundant pathogens neutralizing molecules found in human serum. These antibodies share more than 90% amino acid sequence identity but each subclass has exclusive effector properties including half-life, epitope binding, immunological complex formation, complement activation, triggering of effector cells and placental transport. Moreover, the IgG profile of a given individual is determined by their inherited allotypes that can potentially influence the clinical manifestation of the immune response [122] . However, broadly neutralizing J o u r n a l P r e -p r o o f antibodies (bNAbs) have been found in a rare population of patients that control the infection [123] [124] [125] . These bNAbs tend to target different conserved antigenic regions exposed on the outer surfaces of a pathogen across the circulating strains. Here, in the present study, a protein-protein global docking method (ClusPro server) was used to reveal the shape complementarity between PVs (as ligands) and the interacting domains of antibodies IgG1 and IgG3 (as the receptors) to eliminate the need of a long term exposure of malaria patients to selected antigen mimetics PV1A and PV3B involving the epitopes (B1, B4 and B5) of P.falciparum strains. These antibodies could be considered as bNAbs if they found with a well detectable neutralization activity in wet lab experimental studies [126] [127] [128] [129] [130] . Furthermore, the respective source proteins P28, P25 and MSP1of epitopes B1, B4 and B5 have been characterized as leading vaccine candidates [130, 131] . Also, the antibodies IgG1 and IgG3 have been found associated with human malaria protection [132, 133] . Thus, a structure based vaccinology approach could be exploited to predict the probability of potent PVs that might be able to block infection even more effectively [134] . These data lead to provoke the molecular interaction studies of leading PVs (PV1A as well as PV3B) along with co-crystallized control epitopes towards antibodies IgG1 and IgG3 (Table 7 and Fig. 8 J o u r n a l P r e -p r o o f Molecular dynamics study is crucially for evaluating the stability of the protein-protein complex which can be determined by comparing necessary protein dynamics to their normal modes [136] . The NMA allowed the demonstration of docked protein-protein complex mobility and stabilization. The sequence length of obtained cDNA for PV1A and PV3B were 1092 bp and 1992 bp, correspondingly. The Codon Adaptation Index (CAI) values for PV1A and PV3B were 0.9857 and 0.9584, respectively. For reliable optimization of codon, CAI value should lie between 0.9-1.0 [138] . However, the GC content of improved DNA sequence of PV1A and PV3B were found 42.12% and 43.12% which are lying in the optimal range (30% to 70%) that could be easily expressed in any suitable expression host [139] . Although, P. falciparum antigens could be expressed in E. coli but require the codon harmonization (reduction of amino acid J o u r n a l P r e -p r o o f misincorporation) to improve the immunogenicity [140] . In the present study, the solubilisation probability of recombinant proteins (PV1A and PV3B) to be expressed in E. coli revealed by bioinformatics tools RPSP, Protein-Sol, CamSol and SOLPro was lower compare to positive vaccine controls (C3, C4) that indicated to look for alternative expression host (Table 5) . Additionally, L. lactis was used as expression host alternative to E. coli due to following advantageous properties i) generally recognized as safe (GRAS) microorganism ii) lack of outer membrane (iii) insignificant extracellular proteolysis activity (iv) free of endotoxins (v) no lipopolysaccharide contamination (vii) accommodates cysteine-rich proteins (vii) accessibility of both inducible and constitutive genetic control systems (viii) able to express prone-to-aggregate and/or difficult-to-purify proteins (ix) presentation to the host immune system in the context of microparticles to avoids the immunotolerance which is normally provoked by oral delivery of soluble antigens (x) exhibits similar codon bias to P. falciparum which makes it efficient protein expression and secretion system to outer surface that could easily interact with host immune system [113, [141] [142] [143] . In recent years, several wet lab studies have confirmed the utilization of L. lactis as an expression host to produce properly folded, pure and stable chimeric and/ or single antigenic proteins of many pathogens that elicited high levels of functional antibodies/cytokines including P. [154] . Moreover, L. lactis-mediated delivery of DNA vaccines also lead to the expression of post-translationally modified antigens by host cells resulting in presentation of conformationally restricted epitopes to the immune system for induction of both cellular and humoral immune responses [112] . Also, with the aforementioned properties, the last two decades witnesses the use of genetically engineered L. lactis system as effective oral based vaccine vehicles for delivering antigens of viruses, bacteria and parasites to elicit both systemic and mucosal immunity [155] [156] [157] [158] . Finally, the size of PV1A and PV3B recombinant DNA (obtained after insertion of cDNA into pIL1 expression vector) was observed as 7477 bp and 8377 bp, respectively which lies inside the ORF and could be translated into respective protein sequences with four additional amino acids (MCKC) at the Nterminus (Fig. 10) . Therefore, an ideal multi-epitope polypeptide vaccine should compose of a series of epitopes and or adjuvants that can elicit simultaneous and strong innate and adaptive However, the present problems in the field of multi-epitope vaccine design include the selection of appropriate candidate antigens and systematic arrangement of their immunodominant epitopes for effective oral delivery through virus-like particles and SAPN. The present study successfully utilized the immunoinformatics tools for prediction of suitable epitope ensemble of target proteins for designing a multi-epitope malaria oral vaccine. Surprisingly, so far no licensed malaria vaccine is available in the market to protect worldwide human populations regardless of decades of research. One of the major bottlenecks of malaria vaccine development is immune escape mechanism of pathogen through antigenic variation and/ or HLA diversity. The designed PVs (PV1A and PV3B) under present study may overcome the aforementioned issues as they possess both B and T cell epitopes derived from 5 antigenic proteins that involve multi stages of pathogen life-cycle with worldwide human population coverage (99.46%). Moreover, these PVs have the higher potential to elicit both innate (TLR2 and TLR4) and adaptive (cellular and humoral) immune responses. However, this warrants further experimental validation so as to evaluate their efficacy in the preclinical studies. This study was self-financed and did not receive any grant from funding agency. TLR4 (a, b) , deformability (c, d), eigenvalue (e, f), variance map (g, h), correlation matrix (i, j) and elastic network model (k, l). Coloured bars showed the individual (red) and cumulative (green) variances in the correlation matrix. In the elastic network graph, dots are coloured according to their stiffness, the darker greys indicate stiffer springs and vice versa (m, n). HLA-DRB1*03:08, HLA-DRB1*03:06, HLA-DRB1*03:07, HLA-DRB1*03:09, HLA-DRB1*03:01, HLA-DRB1*03:05, HLA-DRB1*07:03, HLA-DRB1*04:05, HLA-DRB1*08:01, HLA-DRB1*08:17, HLA-DRB1*11:20, HLA-DRB1*08:06, HLA-DRB1*11:01, HLA-DRB1*11:14, HLA-DRB1*08:13, HLA-DRB1*11:07, HLA-DRB1*11:21, HLA-DRB1*11:02, HLA-DRB1*13:21, HLA-DRB1*13:04, HLA-DRB1*13:07, HLA-DRB1*11:28, HLA-DRB1*13:05, HLA-DRB1*13:23, HLA-DRB1*13:01, HLA-DRB1*13:27, HLA-DRB1*13:28, HLA-DRB1*13:22 References 1. 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