A potent multivalent vaccine for modulation of immune system in atherosclerosis: an in silico approach

Atherosclerosis is classically defined as a chronic inflammatory state in which low-density lipoprotein cholesterol (LDL-c) accumulate over intima in medium sized and large arteries [1]. According to the World Health Organization (WHO) reports, cardiovascular diseases are the number one cause of death globally. In particular, cardiovascular diseases account for approximately 40% of all deaths in the world [2]. Hyperlipidemia, smoking, hyperhomocysteinemia, diabetes, and some infectious microorganisms such as cytomegalovirus and Chlamydia pneumonia are known as common risk factors which predispose people for this chronic disorder [3].

Previously, atherosclerosis was assumed merely as a passive aggregation of LDL-c in arterial walls, but recent studies have made this hypothesis more complex and atherosclerosis is now considered as a chronic inflammatory disease [4]. Over the past decade, many investigations have demonstrated that both innate and adaptive immune systems are involved in pathogenesis of atherosclerosis [5]. Many immune cells and molecules such as dendritic cells, B and T lymphocytes, macrophages, neutrophils, and inflammatory cytokines are implicated in initiation and development of atherosclerotic lesions [6].

At the first stages of this disease, high low-density lipoprotein particles in peripheral blood circulation trap in the extracellular matrix of arterial intima. Accumulated LDL-c in sub endothelial layer is converted to oxidized low-density lipoprotein (ox-LDL) through reaction with reactive oxygen species. In vitro and in vivo experiments have shown that classical atherosclerosis risk factors lead to simultaneous expression of adhesion molecules on the surface of endothelial cells. This over expression of adhesion molecules acts as a "danger signal" which activates both the innate immune and the adaptive immune systems. Many immune cells such as monocytes and T cells recruit into the arterial intima and respond to many endogenous and exogenous antigens [7, 8].

Based on above-mentioned evidence, modulation of immune system could represent a useful approach to prevent or treat this disease. Although extensive usage of various drugs such as statin family improve atherosclerosis, these drugs are associated with several side effects [9]. More studies are needed to introduce more effective strategies which confer favorable improvement in atherosclerosis. During last decade, many reports have described the idea of an immune-modulatory approach such as active immunization for improvement of atherosclerosis [10]. The concept of active immunotherapy against atherosclerosis goes back to mid-twenty century when Gero et al. [11] observed that immunization with beta-lipoproteins inhibit progression of atherosclerosis.

One of the most important steps in immunization is to know which antigens are implicated in the disease. As yet, many autoantigens including ox-LDL, β2 glycoprotein I (β-2-GPI), and heat shock protein (HSP) 60 have recognized that play important roles in development of atherosclerosis [12].

ApoB-100, the major protein permanently associated with ox-LDL, has attracted attentions in vaccine researches. Some studies indicated that active immunization using a number of different ApoB-100 peptide sequences such as p2, p143, and p210 led to 40%-70% reduction in development of atherosclerosis [13, 14, 15].

HSPs, a super family of stress proteins classified based on their molecular weight, have been implicated in atherogenesis [16]. Active immunization with various kinds of HSPs have revealed different effects on atherosclerosis. Immunization with HSP65 and their reactive T-cell epitopes was reported to reduce atherosclerotic lesions in different models of experimental atherosclerosis [17, 18]. Also, several studies have stated that immunization with ApoB-100 and HSP60 peptides together exerts synergetic atheroprotective effect [19].

A number of studies demonstrated that β-2-GPI have been involved in the pathogenesis of atherosclerosis [20]. Human β-2-GPI or apolipoprotein H is a plasma protein belongs to the complement control protein superfamily which circulates in the blood at high concentration. This protein has a pivotal role in the binding of anti-phospholipid antibodies (aPL) to negatively charged phospholipids and is identified as an important autoantigen for the development of thromboembolic complications in both arteries and the veins in some autoimmune diseases such as atherosclerosis [21, 22].

Taken the above reports together, both pro-atherogenic and atheroprotective roles for the immune system in atherosclerosis have interested many researchers to design immune-modulatory strategies for active immunization against many of these autoantigens which could probably decrease progression of the atherosclerosis. Since it has been proven that in silico approaches are the best tools for design and evaluation of the vaccines before initiating the experimental study, in this study a new structural model containing three effective autoantigens with antigenic determinants of HSP60, ApoB-100, and β-2-GPI was designed. This chimeric gene was fused together by an appropriate linker and codon optimization for expression in Escherichia coli was carried out. The chimeric protein structure and its ability to induce CD4+ and CD8+ immune responses against these antigens were predicted by in silico approaches.

Atherosclerosis is classically known as a complex inflammatory disorder of arterial intima. Over the past decade, many studies have demonstrated that both protective and pathogenic immune responses are major factors responsible for development of atherosclerosis [5]. Given the significant roles of immune system in development of this vascular disorder, many reports suggested an immune-modulatory strategy such as active immunization to modulate atherosclerosis [37]. Many modalities of vaccine have been used in preclinical studies which represent promising result in modulation of atherosclerosis via active immunization [38]. One of the most important steps in active immunization is recognition of appropriate antigens implicated in the disease. Recent studies have identified many autoantigens such as ox-LDL, β-2-GPI, and HSP60 which play important roles in development of atherosclerosis [12].

Considering that atherosclerosis is a multifactorial disease, it is important to note that blocking of an autoantigen necessarily not to be able modulate atherosclerosis effectively. But this problem could be promote by using multi-component vaccines which are more suitable in preventing and modulating of atherosclerosis in comparison with a mono-subunit vaccine.

In the present study, we selected HSP60, ApoB-100, and β-2-GPI proteins that play pivotal role in development of atherosclerosis. For designing of our multivalent vaccine, we selected the residues 61-361 in the N-terminal region of HSP60 reported to be effective in some immunization studies [4]. Also, the ApoB-100 peptide sequences including p2, p143, and p210 were selected. Recent studies indicated that active immunization using these ApoB-100 peptide sequences leads to 40%-70% reduction in development of atherosclerosis [13, 14]. For the third fragment, the amino acid residues of domain I, II, and V of β-2-GPI protein were selected. This protein is a central antigenic target for the aPL in the pathophysiology of several autoimmune diseases specially atherosclerosis [20, 21]. A logical explanation for this selection is that domains I and II are the best binding sites for the aPL. Also, the structural data suggest a simple membrane binding mechanism in which the positive charges on domain V interact with the negatively charged phospholipids [39, 40]. Since these domains play key roles in pathogenesis of this disease, an immune-modulatory strategy for active immunization against domain I, II, and V could probably be useful for prevention and treatment of this disease by production of specific blocker antibodies against these domains.

The constructed fusion proteins need to the appropriate linkers to bind various parts of these proteins. For efficient separation of the different domains of our chimeric protein, a linker consisting of GGGGs repeats was applied to form a monomeric hydrophobic α-helix and random coil. Our successful experience in using this linker in chimeric gene has shown that it could lead to logically acceptable results. Our selected linker could significantly control the distance and reduce the interference between the domains.

One of the most important steps in designing synthetic genes is codon optimization. We optimized our synthetic gene to enhance recombinant protein production in E. coli. CAI on the optimized gene sequence was 0.66, while wild type gene CAI was only 0.63. This chimeric gene also showed well immunogenic properties in VaxiJen server analysis.

Expression of proteins is significantly dependent on mRNA secondary structure which confer beneficial roles such as regulation of gene expression [41]. Mfold is a common online server usually used to predict RNA secondary structure. The results from mRNA prediction indicated that the mRNA had enough stability for effective translation in the host. Our results showed that the first nucleotides at 5' of mRNAs did not have a hairpin or pseudoknot and the folding free energies of 5'-untranscribed regions were higher than other genomic regions. Therefore, based mentioned results we can expect to increase the translation rates, half-lives, and transcript number of chimeric protein in the host.

Physico-chemical parameters of our chimeric gene were analyzed by ProtParam software. The pI value (pI >7.75) showed basic nature of the protein. Extinction coefficient of chimeric protein at 280 nm was high. Expasy ProtParam classifies the chimeric protein as stable based on the instability index (instability index, 35.59).

Ab initio I-TASSER software was used for predicting three-dimensional structure of the chimeric protein. Our results suggested that ab initio I-TASSER software could predict the folds and good resolution model for our multivalent protein. RMSD and TM score were used to evaluate the reliability and accuracy of predicted models. Expected TM score of 0.55±0.15 validates the accuracy of model. A TM-score more than 0.5 usually indicate the correct topology model and Z-score and C-score were used for showing its confidence. Analysis of chimeric structure based on Ramachandran plot showed a protein with full stability. In Ramachandran plot analysis, approximately an inconsiderable 10.9% of the residues were located in outliner region that could probably be due to the presence of chimeric junctions.

A crucial step in the development of multi-component vaccine based on recombinant protein is to identify B-cell and T-cell epitopes which induce strong B-cell and T-cell responses [42, 43]. In this study, we identified continuous B-cell epitopes of the chimeric protein based on several different features such as antigenicity, accessibility, hydrophobicity, secondary structure, and flexibility analysis. The analysis of all continuous B-cell epitopes was performed completely to obtain good results, without any missing. The identified epitopes on protein surface could interact easily with antibodies, and they were generally flexible [44].

Another important step in computational studies is determination of discontinuous epitopes that are essential for antibody-antigen interaction. DiscoTope which seems to be more reliable than other software recognized 52 conformational epitopes for B cells.

CTLPred server was used for identifying potential T-cell epitopes of chimeric proteins. The selected T-cell epitopes were antigenic with potential to interact with human leukocyte antigen alleles.

In summary, we have applied several bioinformatics methods for immunogenicity prediction of chimeric protein. Our results demonstrated that epitopes of our constructed protein, designed from HSP60, ApoB-100, and β-2-GPI could induce B cell and T cell–mediated immune responses successfully. Therefore, our multivalent vaccine is suggested as a potential candidate for modulation and reduction of atherosclerosis. As the next steps to complete our study on vaccine development, we recommend in vitro synthesis and in vivo experimental studies to evaluate the efficacy of this chimeric vaccine.