Design, structure prediction and molecular dynamics simulation of a fusion construct containing malaria pre-erythrocytic vaccine candidate, PfCelTOS, and human interleukin 2 as adjuvant
© Shamriz and Ofoghi. 2016
Received: 6 October 2015
Accepted: 29 January 2016
Published: 6 February 2016
Malaria infection is still widespread in some parts of the world and threatens the lives of millions of people every year. Vaccines, especially oral vaccines are considered to be effective in reducing the burden of malaria morbidity and mortality. By using recombinant technology, suitable oral hosts could serve as antigen delivering vehicles in developing oral vaccines. This study was aimed towards designing and computational analysis of a fusion protein consisting of Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) fused to human interleukin-2 (IL-2) and M cell-specific peptide ligand (Co1), as a step toward developing a vaccine candidate.
To our best knowledge, the three dimensional (3D) structure of CelTOS is not reported in protein database. Therefore, we carried out computational modeling and simulation in the hope of understanding the properties and structure of PfCelTOS. Then we fused IL-2 to PfCelTOS by a flexible linker and did in silico analysis to confirm the proper folding of each domain in the designed fusion protein. In the last step, Co1 ligand was added to the confirmed fusion structure using a rigid linker and computational analysis was performed to evaluate the final fusion construct. One structure out of five predicted by I-TASSER for PfCelTOS and fusion constructs was selected based on the highest value for C-score. Molecular dynamics (MD) simulation analysis indicated that predicted structures are stable during the simulation. Ramchandran Plot analysis of PfCelTOS and fusion constructs before and after MD simulation also represented that most residues were fallen in favorable regions.
In silico study showed that Co1-(AEEEK)3- IL-2-(GGGGS)3-PfCelTOS construct has a constant structure and the selected linkers are effectively able to separate the domains. Therefore, data reported in this paper represents the first step toward developing of an oral vaccine candidate against malaria infection.
KeywordsMalaria Fusion protein Molecular modeling Molecular dynamics simulations
Infectious diseases are the leading cause of death all over the world [1, 2]. Malaria, also called plasmodium infection, is one of the most important human infectious diseases threatening the lives of millions of people every year. According to WHO malaria report, globally, about 3.2 billion people in 97 countries and territories are at risk of being infected with malaria, and 1.2 billion are at high risk . Although the disease has been eradicated in most areas, it’s still widespread in some regions . The biggest challenges in controlling malaria disease are the emergence of anti-malarial drug resistance and insecticide resistance in parasite and mosquito, respectively [4–6]. Therefore, there is an urgent need to develop novel intervention strategies such as vaccines to reduce the burden of malaria morbidity and mortality. Malaria is most common in poor and developing regions of the world and has a strong negative effect on economic growth . Oral immunogenicity has opened new avenues for the development of vaccines potentially effective in reducing the burden of diseases especially in low-income and developing countries. Oral vaccines are Low cost, easily administered (needle-free), in most cases capable of being stored and transported without refrigeration (Non-Refrigerated) and painless . In contrast to injected vaccines, oral vaccines target mucosal surfaces, which cause stimulation of systemic as well as mucosal immune responses [9–11]. Considering the fact that most pathogens enter the body through mucosal surfaces, mucosal immune system provides the first line of defense against invading bacteria and viruses. Therefore, the simultaneous induction of systemic and mucosal immunity offers an ideal strategy to fight infectious diseases. Despite many advantages that oral vaccines have, only limited numbers of them have been approved for human commercial use and yet significant challenges must be overcome to make oral vaccines closer to reality . One of the challenges is the complexity of mucosal immune system that must discriminate between harmless and dangerous antigens. One way to overcome this problem is to use adjuvants. Oral adjuvants offer exciting possibilities for the formulation of oral vaccines [13–15].
During the past decades considerable progress in recombinant DNA technology has led to the development of fusion proteins. Fusion proteins are novel biomolecules obtained by genetically fusing two or more genes that originally code for separate proteins. Thereby fusion proteins have distinct functions derived from each of their domains. Fusion proteins have wide applications in industry and pharmaceutical protein production.
The general objective of the current study was the design and computational analysis of a fusion protein consisting of Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) fused to human interleukin-2 (IL-2) and M cell-specific peptide ligand (Co1), as a step toward developing a candidate recombinant oral vaccine against malaria. CelTOS is a protein that mediates malarial invasion into its hosts (both vertebrate and mosquito) and is required for effective malaria infection [16–18]. On the other hand, CelTOS amino acid sequence is highly conserved among the Plasmodium species. CelTOS highly conserved amino acid sequence and vital role for malaria infection indicate its important function across all species . Therefore, targeting the immune response to this highly conserved and crucial protein and interfering with its biological function could possibly result in protection against infection by heterologous species of Plasmodium .
In the designed construct, human interleukin 2 (IL-2) is thought to act as mucosal adjuvant. It should be considered that the type of desired immune response (cellular or humoral) will influence the choice of adjuvants for immunization regimens . In case of malaria disease, parasites and infected red blood cells which activate dendritic cells through pattern recognition receptors (PRRs) are phagocytosed and their antigens are presented to T cells. PRR signaling causes the secretion of cytokines that initiate the inflammation that underlies malaria pathogenesis and direct TH1 cell to differentiate. TH1 cells help B cells differentiation and antibody secretion and also secrete IFN-γ, which activates macrophages that phagocytose opsonized parasites and infected red blood cells and subsequently kill them by NO- and O2-dependent pathways. Inflammation induces expression of endothelial adhesion molecules to which infected red blood cells bind. Finally the secretion of anti-inflammatory cytokines from macrophages and regulatory populations of T cells curtailed the Inflammation . To the best of our understanding, adjuvants that increase the TH1 response are more appropriate in malaria vaccine development. Here we focus on human interleukin 2 (IL-2) as mucosal adjuvant. IL-2 has a central role in the cascade of events involved in immune responses and can function as a vaccine adjuvant to increase the immune response to vaccine immunogens [20, 22–26].
IL-2 stimulates T cells to secrete INF-γ. INF-γ is known as immune interferon due to activating macrophages and enhancing phagocytic activity which leads to activation of immune responses. On the other hand, INF-γ results in the expression of MHCI and MHCII molecules on the surface of infected cells and antigen presenting cells, respectively, therefore enhancing antigen presenting and immune system activity. In addition, INF-γ activates the secretion of IgG and its subclasses by effecting on B lymphocytes (The positive effect of INF-γ on immune responses to malaria parasite is discussed above).
Activation of T cells under the influence of IL-2 leads to the secretion of cytokines such as IL-10. Unlike many other interleukins, IL-10 inhibits macrophage activity and secretion of IL-1, IL-12 and TNF. IL-12 acts as inducer for INF-γ and is an effective factor in cellular and innate immune responses against intracellular microbes. Inhibition of IL-12 leads to inhibition of INF-γ production. In fact, IL-10 is well known as the inhibitor of immune responses, especially responses that are set up by macrophages, thereby immune responses are terminated. As mentioned above, the enhancement of inflammation and thereby endothelial adhesion of blood vessels is one of the outcomes of immune system responses to malaria parasite and malaria vaccine. In this case, repressive role of IL-10 prevents excessive inflammation.
Although reported studies have indicated the potential of cytokines as mucosal adjuvants, in order to increase the probability of vaccine candidate binding to intestinal cells, we adopted another strategy. Co1 is a peptide ligand that promotes the binding of ligand-fused antigen to human M-like cells and has also comparable levels of adjuvant activity to Cholera Toxin (CT) . Conjugation of Co1 to the designed construct should result in delivery of antigen to M cells.
Since most of the biological functions of proteins depend upon their 3D structure, in designing multi domain recombinant proteins, proper folding, stability and interaction between domains must be considered. Fusion proteins are much more susceptible to misfolding than single-domain proteins due to the interaction between their different peptide domains. Therefore, in silico analysis of multi domain proteins is an indispensible stage in recombinant protein production projects.
Attempts to model the structure of proteins on the computer began about 30 years ago. Since that time our understanding of protein structure and dynamics has significantly increased and now Protein Data Bank (PDB) contains more than 10,000 high resolution protein structures. Valuable 3D models of a protein that has a clear sequence homology to known proteins can be predicted by homology modeling method. However, even in cases where there is no sequence homology, threading methods relate protein sequences to a library of known structures and predict the likely protein structure. The crystallographic structure of CelTOS has not been reported yet. In the present study, we carried out a molecular modeling study of PfCelTOS protein and designed fusion proteins using iterative threading assembly refinement (I-TASSER) to obtain their 3D structures. Then energy minimization and molecular dynamics (MD) simulation were run to refine the models. The simulations of PfCelTOS and fusion proteins were performed for long time duration of 10 ns and the obtained structures were analyzed to verify further.
Sequence retrieval and fusion protein construction
The amino acid sequences of PfCelTOS, human IL-2 and M cell-targeting ligand, Co1, were retrieved from UniProt (PfCelTOS id: Q53UB8; human IL-2 id: P60568). Amino acid sequences were used to design fusion protein construct. The designed construct consisted of PfCelTOS and human IL-2 mature parts linked together by a flexible linker, whereas Co1 is linked to this construct through a rigid (helical) linker.
Primary structural analysis
Expasy’s Prot Param server  was used to study the physiochemical characters of PfCelTOS and designed fusion constructs such as theoretical isoelectric point (pI), molecular weight, molecular formula, total number of positive and negative residues, instability index , aliphatic index  and grand average hydropathicity (GRAVY) . The instability index provides an estimate of a protein’s stability in vitro. Proteins with instability index smaller than 40 are predicted as stable. A value above 40 indicates that the protein may be unstable.
The aliphatic index of a protein is regarded as a positive factor for the increase of thermostability of globular proteins and is mainly defined as the relative volume occupied by aliphatic side chains (alanine, valine, isoleucine and leucine). The GRAVY score is calculated as the sum of hydropathy values of all the amino acids, divided by the number of residues in the sequence.
Secondary structure prediction
Three-Dimensional (3D) model prediction
The 3D structure of PfCelTOS and fusion proteins were modeled using I-TASSER online server. The raw amino acid sequences of PfCelTOS and fusion proteins were uploaded in FASTA format to I-TASSER server. Tertiary structures were predicted in PDB format. The results generated five top models for each entry which the one with the highest confidence score (c-score) represented the best model and was the structure selected for this study .
A schematic sketch of molecular dynamics procedure
Electrostatic interactions (long-range)
Particle Mesh Ewald (PME)
Total molecular dynamics
Yes/steep = steepest descent minimization
Analysis of simulation
Results and discussion
To our knowledge, the crystallographic structure of CelTOS has not been reported in the protein database. Therefore, in silico analysis of CelTOS 3D structure could be of benefit in predicting its probable structure. Computer prediction and simulation methods can be used to generate representative conformations of a molecule in equilibrium and provide a picture of the way in which a molecule changes from one configuration to another . Therefore, we carried out computational modeling and simulation in the hope of understanding the properties and structure of PfCelTOS. Then we fused IL-2 to PfCelTOS by a flexible linker and did in silico analysis to confirm the proper folding of each domain in the designed fusion protein. In the last step, Co1 ligand was added to the confirmed fusion structure using a rigid linker and computational analysis was performed to evaluate the final fusion construct.
Primary structure analysis
Properties of PfCelTOS, human IL-2 and designed constructs determined by ProtParam
No. of amino acids
Total No. of negatively charged residues (Asp + Glu)
Total No. of positively charged residues (Arg + Lys)
Grand average of hydropathicity (GRAVY)
Secondary structure prediction
Phyre2 prediction and analysis of secondary structure
Three-Dimensional (3D) structure prediction
Prediction of 3D structure was performed by I-TASSER online server using the best aligned template obtained by searching against Protein Data Bank database. I-TASSER (Iterative Threading ASSEmbly Refinement) server (as ‘Zhang-Server’) is a web-based service for the prediction of protein structures and functions. It’s free for academic users and allows them to automatically generate high-quality models of 3D structure of proteins from their amino acid sequences. It detects structure templates from PDB by threading technique. I-TASSER is one of the most successful servers in the community-wide CASP (Critical Assessment of protein Structure Prediction) experiments and was ranked as the No 1 server for protein structure prediction in recent CASP7 CASP8, CASP9, CASP10, and CASP11 experiments.
Structural model refinement
The structural refinement was carried out using molecular dynamics simulation as described in methods. Simulation acts as a bridge between theory and experiment. Indeed we test a theory by conducting a simulation using the same or computationally predicted models and provide a guess at the possible interactions between molecules [39, 42]. Gromacs is a molecular dynamics application developed by Groningen University. Gromacs is able to work in the operating system Linux. The main ability of Gromacs is to perform MD simulation and minimization energy. However, Gromacs does not work alone and should interact with PyMOL and Grace. PyMOL is an application to visualize molecule structure and Grace is an application in Linux to display graphs. Both applications support analysis of MD simulation.
Structure validation of predicted models
The Ramchandran plot analysis of the proteins before and after MD simulation obtained by RAMPAGE Ramachandran Plot Assessment
Comparison of predicted structures before and after MD simulation
The procedure of our study (as shown in Fig. 1) helps to rapid analysis of designed fusion constructs before initializing the recombinant fusion protein lab experiments. As it is obvious, this procedure is fast, inexpensive (since all the servers are free) and simple, especially for inexpert users in this field.
In silico study of Co1-(AEEEK)3- IL-2-(GGGGS)3-PfCelTOS structure through this procedure revealed that designed construction is suitable for fusion protein expression in edible host cells for oral delivery. Flexible linker separates PfCelTOS and IL-2 domains effectively to maintain their proper individual three dimensional structures and allows them to move independently of one another. On the other hand, rigid linker ensures the separation of fusion protein and carrier and lead to the better presentation of fusion construct to human M-like cells. Therefore, data reported in this paper represents the first step toward developing of an oral vaccine candidate against malaria infection.
The authors would like to thank Dr. Mohammad Reza Khoramizadeh for his good immunological advice and also thank to Mr. Hojat Borna for his help in computational techniques.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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