Chapter 1 Structural Immunoinformatics on Modeling Epitope Variability and Agretope
Stability
Immune system is the defense mechanism of our body against infectious agents and other foreign organisms in rather complicated process. Immunoinformatics is the computational method focusing on immune-related interactions in consists of immune-related databases, epitope prediction, vaccine design, and so forth.
In the past, vaccine development depends on biochemical and immunological experiments, such as attenuation of the wild type pathogens by random mutations and serial passages, X-ray crystallography studies of antibody/antigen structure, phage display library, overlapping peptides, NMR, radioimmunoassay, immunofluorescence, ELISA, Western blotting, and immunohistochemistry, which is very expensive, time-consuming, with low immunogenicity and reversible.[1] In recent, high-throughput experiment and computational advance progresses the understanding on immune system greatly. Assisted with epitope prediction approach, we can reduce the spectrum of dry lab target proteins and reduce the cost of wet lab experiments.
1.1 Background Review
1.1.1 Nasopharyngeal carcinoma (NPC)
Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma that occurs on the epithelium of nasopharynx.[2] It is a common malignancy in south-east Asia countries including Taiwan, Indonesia, Singapore, Malaysia, and Vietnam in addition to Hong Kong and southern China.[3] Environmental factors, Epstein-Barr virus (EBV), and genetic susceptibility are thought to play important roles towards the development of NPC.
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The radiotherapy or concurrent chemoradiotherapy of NPC clinical treatment may still occur local pathologic failure and distant metastasis in many patients despite of some outcome improvements. Moreover, the radiotherapy with chemotherapy often accompanies with acute side effects and long-term sequelae including secondary malignancy.[3] Pursue for novel approaches aiming at improving outcome and reducing demand for conventional cytotoxic therapy seems thus to indicate immunotherapy as of an attractive option under development. The crucial advantage of antigen-specific immunotherapy is the ability to evaluate and monitor immune responses against targeted antigens and to correlate the findings with clinical responses.
1.1.2 Epstein-Barr virus (EBV)
Epstein-Barr virus (EBV) is a member of the herpesvirus family.[4] It has a double-stranded DNA genome of 184-kb pairs in length, encoding nearly 100 proteins.[5] It was the first virus to be associated to human cancer. EBV attack B-lymphocyte as primary target, resulting in lifelong infection.[5] Presence of EBV genome is demonstrated virtually in most NPC cells through oncogenesis process of EBV latent infections. Regardless of geographical origin, EBV is uniformly detected in patients with undifferentiated and poorly-differentiated NPC.[6]
The EBV-NPC oncogenesis process may equip both proliferation advantage and immune evasion in order to overcome efficient anti-EBV immune clearance mechanisms of antibody-mediated immunity (AMI) of antibody-dependent cell-mediated cytotoxicity (ADCC) as well as cell-mediated immunity (CMI) of cytotoxic T lymphocyte (CTL)-initiative cytotoxic apoptosis during either latent and/or regular EBV infection phases.
In spite of being a latent infection in B cells, inhibition by a population of EBV-specific CTLs was observed.[7] Both in vitro and in vivo, these CTLs have been shown to have potent antiviral activity. Growing evidence revealed that cytotoxic T lymphocytes-based immunotherapy is effective in other EBV-linked malignancies, such as post-transplant lymphoproliferative disorders (PTLD). The success of this therapy has encouraged researchers to develop similar strategies for other EBV-positive tumors, such as NPC.
1.1.3 Latent membrane protein 1 and 2 (LMP1/LMP2)
Notably, NPC latent infection case expresses only limited EBV viral antigens with less immunogenicity including EBV-encoded nuclear antigen (EBNA1) and latent membrane protein 1 and 2 (LMP1 & LMP2) which is greatly unlike that regular EBV latent infection
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case with expression of many EBV viral antigens in symptomatic EBV-related diseases.[8]
Both LMP1 and LMP2 may serve potentially as better vaccine targets due to the poor processing efficiency over with EBNA antigen while in antigen-presenting cells (APC) as of the infected B lymphocytes according to literatures in murine models.[9]
However, LMP1 and LMP2 are with main shortages both in strong oncogenicity and as well in weak immunogenicity by stringent class I major histocompatibility complex (MHC) presentation in the host cell of infected B lymphocytes in order for cytotoxic T lymphocyte (CTL) activations due to that as a result may shift balance towards flexible MHC-II presentation of infected B lymphocytes in order for T helper (Th) lymphocyte activations with subtle feedback network to enhance B lymphocyte proliferations towards aberrant tumorigenesis.[10] Specifically, the basic proliferation advantage is likely from encoding EBV latent infection membrane protein 1 (LMP1) with growth factor receptor-like activity and as well the critical immune evasion is likely from ethnic class I human leukocyte antigen (HLA1) difference with mutating EBV genome for poor immunogenicity responses at AMI-antigen epitopes and CMI-antigen epitopes/agretopes within LMP1/LMP2 and/or EBNA of EBV-encoded proteins.
1.1.4 Epitope variability and agretope stability
Epitope is a part of a protein antigen recognized by either a particular antibody molecule or a particular T-cell/B-cell receptor of the immune system.[12] On the other hand, agretope is histocompatibility complex (MHC) binding motif of a protein antigen.[13] Highly likely, the EBV-NPC immune evasion on ultimatum agretope mutant of CMI maybe the most crucial strategy for oncogenic negative selection against which the host immune system cannot counter-act efficiently as opposed to that epitope mutants of AMI and CMI in oncogenic cells maybe eventually removed with affinity maturations of B cell receptor (BCR) and T cell receptor (TCR) by means of hyper mutations with gene rearrangements during the long-term process of EBV-related NPC oncogenesis.
1.1.5 Immune evasion
Modulation of T-cell recognition is of crucial importance for EBV, because this herpesvirus resides intracellularly for most of its life cycle. During the latent and lytic phase of EBV infection, antigen presentation of host cell via MHC class I and class II is blocked by multiple EBV gene products. Detection of cells harboring latent and replicating EBV by CD8+ and
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CD4+ T lymphocytes is thus prevented. These so-called immune-evasive maneuvers prevent the induction of programmed cell death and develop persistent infection and tumor growth eventually. The NPC immune evasion of agretope maybe exemplified with the prevalence difference on ethnic HLA1 spectrum along with EBV-LMP1 mutant assorts. The A*02:07 (common in Taiwan population) shows higher prevalence of EBV-NPC than A*02:01 (common in Caucasian population) while further with additional synergistic B*4601/B*14 and extended haplotype HLA A*3303- B*5801/2- DRB1*0301- DQB1*0201/2- DPB1*0401.
The EBV-NPC biopsies from Taiwan cases reveal variant NPC-related LMP1 (NLMP1) {GenBank: X66863} of immune evasion which shares high amino acid sequence homology prominently with prototype B95.8-LMP1 {GenBank: V01555} and CAO-LMP1 in China population. Importantly, NLMP1 over-expression in Balb/c class I major histocompatibility complex (MHC1)-context towards regressing experimental murine EBV-NPC may be resulted likely from regaining strong agretope presentation of NLMP1 in mice MHC1 context in disregard of selected immune evasion of original NLMP1 in tumor microenvironment of human HLA1 context that was with weak agretope binding in CMI antigen presentation and with immune suppression in local immune suppressive cells. The ethnic A*02:07 difference of genetic susceptibility to EBV-NPC may indicate that omega-shape NLMP1np in CMI antigen presentation is required for crucial docking onto A*02:07 pit while with overlooked under-side agretope of head-anchor and tail-anchor along with overstressed bulge-side epitope in order for inducing adequate immunogenicity presentation towards effective CMI-CTL induction.
1.1.6 Cancer immunotherapy
Despite recent treatment advances that have improved the quality of life of patients with nasopharyngeal cancer, local regional failure and distant metastasis still occur in many patients. Innovative therapies are therefore still under developed. Immunotherapy is an attractive therapeutic option. There are several advantages to make use of the immune system to fight cancer. First, the immune system has the natural ability to specifically identify and kill neoplastic cells while sparing normal tissue. Second, the immune system demonstrates potential to evolve with the cancer cells. Both humoral and cellular immune system involve with cells with a vast array of clonally distributed antigen receptors. The diversity of these receptors enables the immune system to recognize foreign and/or altered antigens and to discriminate self, or normal cells, from non-self, or cancerous cells.[11]
The immunotherapeutic regime against EBV-NPC for instance may conveniently exploit various aspects including AMI-ADCC with vaccine peptides, CMI-CTL with DNA vaccines,
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and microenvironment immune suppression with in vitro cell activation towards in vivo adoptive cell transfer. The current challenges except vaccine peptides still install obstacles including weak HLA1-binding agretope in host cell or dendritic cell, and specific delivery of DNA vaccine to host cells without damaging innocent bystander cells. Immunoinformatics is with remarkably high practical potential in feasible application of epitope/agretope binders onto AMI-BCR and CMI-HLA/TCR towards mining putative anchor modified agretope (Ama) and agretope complex enhancer (Ace) with reinforced binding affinity (BAff) of NLMP1 agretope and A*02:07 pit in order to likely improve NPC-CMI specifically while with low adverse cytotoxic effect due to non-specificity.
In this study, we implement bio-mimicry peptide design algorithm (bmPDA) comprising peptide database construction of building blocks, peptide backbone modeling of building block candidates, and quality evaluation on predicted nona-peptide structures. Our bmPDA of structure-based immunoinformatic approach aims at designing EBV immunogenicity-related omega-shape NLMP1 nona-peptide (NLMP1np) structures. We apply in-house bmPDA-tool towards applications of predicting A*02:07-binding EBV-NLMP1np structures in order that the verification on putative epitope and agretope quality may be accomplished with outsourcing tools of NetCTL server and Molegro Virtual Docker (MVD) software. The BAff with designed omega-shape NLMP1np and LMP1np structures on docking both HLA pits of A*02:07 {PDB: 3OXS} and A*02:01 {PDB: 1BD2} may be evaluated with MDV tool towards mining putative Ama and Ace candidates among which may be identified in modified-anchor assorts and FDA-approval drugs based on stable BAff of NLMP1np agretope and A*02:07 pit in order to specifically improve NPC CMI yet likely with low adverse effect due to non-specificity.