Prediksi Efektivitas Interaksi antara Antibodi dan Vaksin H1n1 melalui Metode Molecular Docking secara In Silico

Flu babi H1N1 merupakan penyakit menular akibat virus influenza tipe A yang telah menjadi pandemik dan mortalitasnya sangat tinggi pada manusia. Pemberian vaksin menjadi salah satu upaya pencegahan penyakit tersebut. Bagian antigenik (epitope) virus H1N1 digunakan untuk merancang suatu vaksin. Beberapa epitope telah diprediksi dari perwakilan protein hemagglutinin (HA), neuramidase (NA), dan matriks 2 (M2) virus H1N1. Pendekatan in silico dilakukan melalui kombinasi prediksi antigen pada tahapan respons imun, yaitu proteasomal cleavage (NetChop), Transporter Antigen Processing (TAP) binding (TAPPred), dan Major Histocompability Complex (MHCPred). Upaya meningkatkan respons imun juga dilakukan dengan memprediksi epitope sel B menggunakan server DiscoTope (conformational epitope) dan BepiPred (sequensial epitope). Enam model vaksin, yaitu NHM, MHN, HNM, MNH, HMN, dan NMH diperoleh dari 21 kombinasi terbaik epitope sel T dan sel B sebagai representasi variasi allele Human Leukocyte Antigen (HLA) dan protein virus H1N1 sehingga diharapkan mampu memberikan respons imun. Struktur 3D vaksin diprediksi dan dimodeling menggunakan server CPHModels dan program Swiss-Pdb Viewer (Deep View). Hasil struktur 3D vaksin dievaluasi menggunakan Ramachandran Plot, BLASTp (database PDB virus), dan FeatureMap 3D. Vaksin terdiri dari 258 asam amino dan memiliki lebih dari 50 % kesamaan struktur 3D dengan protein dalam database. Proses akhir efektivitas antibodi terhadap vaksin diuji melalui molecular docking antara vaksin dengan antibodi dalam database, diperoleh 14 clustering dengan waktu yang dibutuhkan sekitar 18 detik dan data energi minimum interaksi didapatkan antara antibodi terhadap vaksin NHM sebesar -13,6859 kkal/mol

Development of peptide vaccines in dengue

Dengue is one of the most important arboviral infection worldwide, infecting up to 390 million people and causing 25,000 deaths annually. Although a licensed dengue vaccine is available, it is not efficacious against dengue serotypes that infect people living in South East Asia, where dengue is an endemic disease. Hence, there is an urgent need to develop an efficient dengue vaccine for this region. Data from different clinical trials indicate that a successful dengue vaccine must elicit both neutralizing antibodies and cell mediated immunity. This can be achieved by designing a multi-epitope peptide vaccine comprising B, CD8+ and CD4+ T cell epitopes. As recognition of T cell epitopes are restricted by human leukocyte antigens (HLA), T cell epitopes which are able to recognize several major HLAs will be preferentially included in the vaccine design. While peptide vaccines are safe, biocompatible and cost-effective, it is poorly immunogenic. Strategies to improve its immunogenicity by the use of long peptides, adjuvants and nanoparticle delivery mechanisms are discusse

Computer-Aided vaccine design for selected positive-sense single stranded RNA viruses

Spontaneous mutations and lack of replication fidelity in positive-sense single stranded RNA viruses (+ssRNA virus) result in emergence of genetic variants with diverse viral morphogenesis and surface proteins that affect its antigenicity. This high mutability in +ssRNA viruses has induced antiviral drug resistance and ability to overcome vaccines that subsequently resulted in rapid viral evolution and high mortality rate in human and livestock. Computer aided vaccine design and immunoinformatics play a crucial role in expediting the vaccine production protocols, antibody production and identifying suitable immunogenic regions or epitopes from the genome sequences of the pathogens. T cell and B cell epitopes can be identified in pathogens by immunoinformatics algorithms and methods that enhance the analysis of protective immunity, vaccine safety, immunity modelling and vaccine efficacy. This rapid and cost-effective computational vaccine design promotes development of potential vaccine that could induce immune response in host against rapidly mutating pathogens like +ssRNA viruses. Epitope-based vaccine is a striking concept that has been widely employed in recent years to construct vaccines targeting rapidly mutating +ssRNA viruses. Therefore, the present review provides an overview about the current progress and methodology in computer-aided vaccine design for the most notable +ssRNA viruses namely Hepatitis C virus, Dengue virus, Chikungunya virus and Coronaviruses. This review also highlights the applications of various immunoinformatics tools for vaccine design and for modelling immune response against +ssRNA viruses

In Silico Modeling and Immunoinformatics Probing Disclose the Epitope Based PeptideVaccine Against Zika Virus Envelope Glycoprotein

Zika virus (ZIKV) is an aedes mosquito borne pathogen belonging to the member of flaviviridae subgroup is the causative agent of an emerging disease called Zika fever, known as a benign infection usually presenting as influenza like illness with cutaneous rash. Due to recent epidemic outbreaks it is realized as a major health risk which need enhanced surveillance, but no attempt has been made to design an epitope based peptide vaccine against Zika virus. Viral envelope proteins are derived from host cell membrane proteins with some viral glycoproteins and are used to cover their protective protein capsid, help the viruses to enter host cells and help them to avoid the host immune response. In this study, amino acid sequence of ZIKV envelope glycoprotein was obtained from a protein database and examined with in silico approaches to determine the most immunogenic epitopes for B cell and T cell which could induce humoral as well as cell mediated immune response. Both the linear and conformational epitopes for B cell were predicted by immunoinformatics tools housed in IEDB resources. The peptide sequence DAHAKRQTVVVLGSQEGAV from position 121 and peptide sequence from 117-137 amino acids were predicted as most potential B cell linear and conformational epitopes respectively. Epitopes for CD4+ and CD8+ T cell were also predicted by using tools within IEDB resource and peptide sequence MMLELDPPF from position 250-258 amino acids was predicted as most immunogenic CD8+ T cell epitope with immune response evoking ability prediction score (I pMHC) of 0.09139 and conservancy of 52.17%. The innate immune response for ZIKV envelope glycoprotein was determined by interferon (IFN)-gamma effectuation and mimicking capacity by immunoinformatics and molecular docking study respectively. However, this is an introductory approach to design an epitope based peptide vaccine against Zika virus; we hope this model will be very much helpful in designing and predicting novel vaccine candidate

Insights to Protein Pathogenicity from the Lens of Protein Evolution

As protein sequences evolve, differences in selective constraints may lead to outcomes ranging from sequence conservation to structural and functional divergence. Evolutionary protein family analysis can illuminate which protein regions are likely to diverge or remain conserved in sequence, structure, and function. Moreover, nonsynonymous mutations in pathogens may result in the emergence of protein regions that affect the behavior of pathogenic proteins within a host and host response. I aimed to gain insight on pathogenic proteins from cancer and viruses using an evolutionary perspective. First, I examined p53, a conformationally flexible, multifunctional protein mutated in ~50% of human cancers. Multifunctional proteins may experience rapid sequence divergence given trade-offs between functions, while proteins with important functions may be more constrained. How, then, does a protein like p53 evolve? I assessed the evolutionary dynamics of structural and regulatory properties in the p53 family, revealing paralog-specific patterns of functional divergence. I also studied flaviviruses, like Dengue and Zika virus, whose conformational flexibility contributes to antibody-dependent enhancement (ADE). ADE has long complicated vaccine development for these viruses, making antiviral drug development an attractive alternative. I identified fitness-critical sites conserved in sequence and structure in the proteome of flaviviruses with the potential to act as broadly neutralizing antiviral drug target sites. I later developed Epitopedia, a computational method for epitope-based prediction of molecular mimicry. Molecular mimicry occurs when regions of antigenic proteins resemble protein regions from the host or other pathogens, leading to antibody cross-reactivity at these sites which can result in autoimmunity or have a protective effect. I applied Epitopedia to the antigenic Spike protein from SARS-CoV-2, the causative agent of COVID-19. Molecular mimicry may explain the varied symptoms and outcomes seen in COVID-19 patients. I found instances of molecular mimicry in Spike associated with COVID-19-related blood-clotting disorders and cardiac disease, with implications on disease treatment and vaccine design

Computational immunology : analyses of viral escape, epitope binding and T cell receptor recognition

It has been shown repeatedly that infectious diseases in humans have strong associations with the human leukocyte antigen system, but an understanding of the basis of these associations remains elusive. Adaptive immune responses involving CD4 and CD8 T lymphocytes are dependent on (1) the appropriate and effective processing of a peptide from a protein source, (2) the stable binding of the peptide to the HLA molecule and (3) the recognition of this complex by the T cell receptor. In this thesis, we present work helping to better define such host-virus dynamics, examining aspects relating to each of the described steps. We examined two large patient cohorts, the first infected with HIV-1 and the second with HCV. We identified viral escape mutations and thus potential immune epitopes. Also, we examined the possible effects of HLA genotypes on the development of drug resistance mutations (HIV-1) and the success of antiviral therapy (HCV). To better understand the stable binding of peptides to HLA molecules, we evaluated the performance of diverse HLA class I prediction methods on large datasets, showing that all leading methods are capable of good to excellent performance. Finally, we developed the first algorithms, based on the interactions found in actual experimental structures, which allow for the prediction of interactions between residues in the T cell receptor\u27s CDR loops and residues in the HLA-peptide antigen. The algorithms had good performance under cross-validation.Wiederholt wurden viele Zusammenhänge menschlicher Infektionskrankheiten mit dem Human-Leukozyten-Antigen-System aufgezeigt, doch ein vollständiges Verständnis dieser Zusammenhänge fehlt. Adaptive Immunantworten mit CD4- und CD8-T-Lymphozyten sind abhängig von (1) einer angemessenen und effektiven Bearbeitung eines Peptids, (2) der stabilen Bindung des Peptids an das HLA-Molekül und (3) der Erkennung dieses HLA-Peptid-Komplexes durch den T-Zell-Rezeptor. In dieser Dissertation präsentieren wir Arbeiten, die helfen, diese Wirt-Virus-Dynamik besser zu definieren, indem wir Aspekte jedes dieser beschriebenen Schritte untersuchen. In zwei großen Patientengruppen (die erste mit HIV-1 und die zweite mit HCV infiziert) identifizierten wir virale Escape-Mutationen und damit potentielle Immun-Epitope. Wir untersuchten die möglichen Auswirkungen des HLA-Genotypes auf die Entwicklung von Resistenz-Mutationen (HIV-1) und den Erfolg einer antiviralen Therapie (HCV). Um die stabile Bindung von Peptiden an HLA-Moleküle besser zu verstehen, untersuchten wir die Leistung verschiedener HLA-Klasse I-Prognoseverfahren und zeigten, dass alle führenden Methoden gute bis sehr gute Ergebnisse liefern können. Abschließend haben wir die ersten Algorithmen entwickelt, die die Interaktionen zwischen den Aminosäuren der CDR-Schleifen des T-Zell-Rezeptors und Aminosäuren des HLA-Peptid-Komplexes vorhersagen. Diese Algorithmen zeigten gute Leistung unter Cross-Validierung

CD8+ T Cell Serotype-Cross-Reactivity is a Predominant Feature of Dengue Virus Infections in Humans: A Dissertation

The four serotypes of dengue virus (DENV 1-4) have a significant and growing impact on global health. Dengue disease encompasses a wide range of clinical symptoms, usually presenting as an uncomplicated febrile illness lasting 5-7 days; however, a small percentage of infections are associated with plasma leakage and bleeding tendency (called dengue hemorrhagic fever, DHF), which can result in shock. Epidemiological studies indicate that severe dengue disease most often occurs during secondary heterotypic DENV infection. Additionally, plasma leakage (the hallmark of DHF) coincides with defervescence and viral clearance, suggesting that severe disease arises from the immune response to infection rather than a direct effect of the virus. A number of studies have found increased levels of markers of immune cell activation in patients with DHF compared to patients with the less severe form of disease (DF). These markers include IFNγ, TNFα, soluble CD8, soluble IL-2 receptor, soluble TNF receptor, and CD69, which support a role for T cells in mediating immunopathology. Because of the high homology of DENV 1-4, some degree of serotype-cross-reactivity is seen for most T cell epitopes. A high percentage of DENV-specific T cells recognize multiple DENV serotypes, as demonstrated by peptide-MHC (pMHC) tetramer binding and in vitro functional assays performed on PBMC from subjects vaccinated with an experimental DENV vaccine or naturally-infected subjects with secondary (\u3e1) DENV infection. This thesis sought to address several gaps in the literature, specifically whether T cell responses differ in primary versus secondary (natural) infection. We studied the frequency, phenotype, and function of DENV-specific T cells. We demonstrated substantial serotype-cross-reactivity of antigen-specific T cells generated in response to naturally-acquired primary as well as secondary DENV infection. The frequency of A11-NS3133 epitope-specific T cells during acute infection did not correlate with disease severity. However, the peak frequency occurred earlier in primary infection while the frequency of CD45RA+ T cells declined quicker in secondary infection, suggesting the expansion of DENV-specific memory T cells. DENV-immune T cells exhibited different functional capabilities that were dependent on the particular serotype of infection. Specifically, DENV-1 or -3 stimulation of A11-NS3133 epitope-specific T cell lines resulted in robust function that included IFNγ production, whereas DENV-2 stimulation resulted in limited function that often included MIP-1β but not IFNγ production. These data support a role for T cells in DENV infection and offer new insights into their potential contribution to dengue pathology

HLA class I supertype and supermotif definition by chemometric approaches.

Activation of cytotoxic T cells in human requires specific binding of antigenic peptides to human leukocyte antigen (HLA) molecules. HLA is the most polymorphic protein in the human body, currently 1814 different alleles collected in the HLA sequence database at the European Bioinformatics Institute. Most of the HLA molecules recognise different peptides. Also, some peptides can be recognised by several of HLA molecules. In the present project, all available class I HLA alleles are classified into supertypes. Super - binding motifs for peptides binding to some supertypes are defined where binding data are available. A variety of chemometric techniques are used in the project, including 2D and 3D QSAR techniques and different variable selection methods like SIMCA, GOLPE and genetic algorithm. Principal component analysis combined with molecular interaction fields calculation by the program GRID is used in the class I HLA classification. This thesis defines an HLA-A3 supermotif using two QSAR methods: the 3D-QSAR method CoMSIA, and a recently developed 2D-QSAR method, which is named the additive method. Four alleles with high phenotype frequency were included in the study: HLA-A*0301, HLA-A*1101, HLA-A*3101 and HLA- A*6801. An A*020T binding motif is also defined using amino acid descriptors and variable selection methods. Novel peptides have been designed according to the motifs and the binding affinity is tested experimentally. The results of the additive method are used in the online server, MHCPred, to predict binding affinity of unknown peptides. In HLA classification, the HLA-A, B and C molecules are classified into supertypes separately. A total of eight supertypes are observed for class I HLA, including A2, A3, A24, B7, B27, B44, CI and C4 supertype. Using the HLA classification, any newly discovered class I HLA molecule can be grouped into a supertype easily, thus simplifying the experimental function characterisation process

Single-round rhabdovirus replicons and an augmented RBD: A safe and effective combination for a SARS-CoV-2 vaccine

The ongoing SARS-CoV-2 pandemic can only be curbed by a concerted, global vaccination effort. Vaccines are used in healthy populations and represent a voluntary, preventative intervention while requiring application in most of the populace to induce herd immunity. Therefore, vaccines must meet the highest possible safety standards and at the same time induce a beneficial immune reaction and protection against the pathogen in question. To address these functionally opposite goals, we designed, created, and tested a vaccine platform based on non spreading, single round rhabdovirus vectors expressing a highly immunogenic antigen construct consisting of a cell surface anchored SARS CoV 2 receptor binding domain (RBD) that, in addition to being presented on the cell surface of transduced cells, is incorporated into budding rhabdovirus virions and non infectious pseudovirus particles. The RBD sequence was derived from the ancestral SARS CoV 2 Wuhan strain and genetically fused to the RABV G stem, transmembrane domain, and intracellular tail. This so termed “minispike” was characterized in detail, revealing efficient expression, post translational modification, and insertion into cell membrane and viral particles. Correct folding and adoption of a biologically relevant conformation was demonstrated by specific recognition of the construct by COVID 19 patient sera and SARS CoV 2 S binding mAbs on both live and fixed cells. A series of G deleted, single round vectors containing one to three copies of the minispike cistron was cloned, rescued and characterized in regard to minispike expression levels and viral titers. We then chose VSV∆G minispike eGFP (monovalent, non-spreading) to immunize BALB/c mice. Sera from these mice was tested for the capacity to neutralize SARS CoV 2 S mediated infection in authentic and surrogate virus neutralization assays. Considerable neutralization titers comparable to convalescents from severe COVID 19 were induced already after prime vaccination and further improved by boost vaccination. The minispike immune sera were further tested for their neutralization capacity against prevalent SARS CoV 2 variants of concern (including alpha, beta, gamma and delta), displaying a remarkable resistance to escape. The delta variant showed the most severe reduction in neutralizing titers (8 to 12 fold). In contrast, the beta variant, which is described to have the most pronounced immune escape phenotype for both convalescents and vaccinees, was readily neutralized with only a two fold reduction in neutralizing titers. These findings indicate the induction of a diverse and robust neutralizing antibody response targeting multiple distinct epitopes on the S RBD by minispike immunization. Finally, live virus challenge experiments in susceptible K18-hACE mice with ancestral SARS CoV 2 as well as the delta variant revealed that a single vaccination with VSV∆G minispike eGFP is sufficient to completely protect the mice from all clinical signs of SARS CoV 2 induced disease. Remarkably, even though we saw the highest decline in neutralizing titers against delta, mice were still equally well protected against this variant.Die anhaltende SARS-CoV-2-Pandemie kann nur durch konzertierte, weltweite Impfaktionen eingedämmt werden. Impfstoffe gegen pandemische Erreger stellen eine freiwillige, vorbeugende Prävention am Gesunden dar und erfordern Anwendung bei dem Großteil der Bevölkerung, um eine effektive Herdenimmunität herbeizuführen zu können. Daher müssen Impfstoffe höchstmögliche Sicherheitsstandards erfüllen und gleichzeitig eine effektive, spezifische Immunreaktion und einen verlässlichen Schutz gegen den jeweiligen Erreger auslösen. Um diese funktionell gegensätzlichen Ziele zu erreichen, haben wir eine Impfstoffplattform entwickelt und getestet, die auf sich nicht ausbreitenden, sog. „single-round“ Rhabdovirus-Vektoren basiert, welche ein hoch immunogenes Antigenkonstrukt exprimieren. Dieses besteht aus der zelloberflächenverankerten Rezeptorbindungsdomäne (RBD) des SARS-CoV-2 Spike (S) Proteins, die sowohl auf der Zellmembran transduzierter Zellen als auch in Rhabdovirus-Virionen und nicht-infektiösen Pseudovirus-Partikel eingebaut und präsentiert wird. Die RBD-Sequenz wurde vom ursprünglichen SARS-CoV-2-Wuhan-Stamm abgeleitet und genetisch mit dem extrazellulären Stamm, der Transmembran- und Intrazellulärdomäne des Tollwutvirus-Glykoproteins (G) fusioniert. Dieses sogenannte „Minispike“ wurde im Detail charakterisiert, wobei effiziente Expression, posttranslationale Modifikation und Insertion in Zellmembranen und Viruspartikel gezeigt werden konnten. Die korrekte Faltung in eine biologisch relevante Konformation wurde durch die spezifische Erkennung des Konstrukts durch COVID-19-Patientenseren und SARS-CoV 2 S-bindende monoklonale Antikörper sowohl in Lebendzell-Mikroskopie als auch auf fixierten Zellen bestätigt. Eine Reihe nicht ausbreitungsfähiger, „single-round“ rekombinanter Viren, bei denen das Gen für das virale Glykoprotein gegen eine bis drei Kopien des Minispike-Gens ausgetauscht war, wurden im Hinblick auf Expressionslevel des Minispike-Proteins und Attenuierung charakterisiert. Anschließend wurden BALB/c-Mäuse mit einem Konstrukt immunisiert, welches auf dem G-deletierten Vektorvirus VSV basiert und eine Kopie des Minispike-Gens (VSV∆G minispike eGFP) aufweist. Immunseren von diesen Mäusen wurden auf ihre Fähigkeit getestet, eine SARS-CoV 2 S-vermittelte Infektion zu neutralisieren, wobei sowohl S-pseudotypsierte Pseudoviren als auch authentisches SARS-CoV-2 zum Einsatz kamen. Eine erhebliche Neutralisationsaktivität, vergleichbar mit denen schwerkranker COVID-19 Patienten, wurde bereits nach der Grundimpfung induziert und durch eine Auffrischimpfung weiter erhöht. Diese Minispike-Immunseren wurden weiter auf ihre Neutralisationskapazität gegen besorgniserregende vorherrschende SARS-CoV-2-Varianten (einschließlich Alpha, Beta, Gamma und Delta) getestet und zeigten eine bemerkenswerte Resistenz gegen Immunevasion. Die Delta-Variante wies dabei mit einer 8- bis 12-fachen Reduktion der Neutralisationstiter den höchsten Immun-Escape auf. Im Gegensatz dazu wurde die Beta-Variante, für die sowohl bei Genesenen als auch bei Geimpften der ausgeprägteste Immun-Escape-Phänotyp beschrieben wurde, hocheffektiv (mit einer Reduktion um 50%) neutralisiert. Diese Ergebnisse sprechen für die Induktion einer breiten und robusten neutralisierenden Antikörperantwort nach VSV∆G Minispike-Immunisierung, die auf mehrere, unabhängige Epitope der S RBD abzielt. Diese Ergebnisse wurden in SARS-CoV-2 Challenge Experimenten in K18-hACE-Mäusen bestätigt. Sowohl bei Challenge mit dem ursprünglichen SARS-CoV 2 als auch mit der Delta-Variante reichte eine einzelne Impfung mit VSV∆G minispike eGFP aus, um alle Mäuse vollständig vor sämtlichen klinischen Anzeichen einer SARS CoV 2 induzierten Erkrankung zu schützen. Besonders hervorzuheben ist dabei, dass die Mäuse gegen die Delta-Variante ebenso gut geschützt waren, wie gegen das parentale Virus, obwohl die Neutralisationstiter gegen diese Variante die höchste Reduktion aufwiesen. Dies unterstreicht die Robustheit eines durch eine einzelne Impfung mit VSV∆G Minispike eGFP hervorgerufene Schutzwirkung