Marco Papageorgiou

An inter-disciplinary approach to developing a vaccine against Dengue virus: using multiepitope MHC-peptides, CRISPR/ cas9 technology, and a Lactic acid bacteria (LAB) delivery system. CONTENTS 1.0 Introduction……………………………………………………………………………………….1-2 2.0 Approach to Research……………………………………………………………………………..2-3 3.0 Methodology……………………………………………………………………………………….3 3.1 Work-flow breakdown……………………………………………………………………..4 4.0 Experimental Design………………………………………………………………………………5 4.1 Multiple-epitope design: Immunoproteomics and Immunoinformatics…………………5-6 4.2 CRISPR/Cas9: components and incorporation into a LAB system………………………7 4.3 Applying LAB in peptide-MHC surface display…………………………………………8 4.3.1 Using the Nisin—cotnrolled gene expression system (NICE)…………………9 4.4 3D Cell Culturing………………………………………………………………………..10 4.4.1 Training bioinformatics programs from 3D cell culturing datasets……………11 4.5 Creating a promising Dengue vaccine candidate combining all approaches…………..11-12 5.0 Justification of study…………………………………………………………………………..13-14 6.0 Motivations of study…………………………………………………………………………….15 6.1 Research-based………………………………………………………………………..15-16 6.2 Administrative-based………………………………………………………………….16-17 Bibliography………………………………………………………………………………………18-19 !i 1.0 INTRODUCTION Research into human diseases and viruses will continue to be on ongoing pursuit within the world of scientific inquiry. From the onset of the information age, science has taken a dramatic turn into digitised forms of data; i.e. accumulative systems biology of many biological processes in vivo. One stand out feature of this shift to a more holistic, biotechnological, ‘omics’ and bioinformatics approach is the ability for researchers to better understand the relationships between protein networks and subsequent phenotype related to an organism, and its associated modes of functioning; i.e. microorganisms and infectious diseases. This holistic approach to biological research can be perfectly aligned to those diseases which are currently in endemic classification; for example dengue virus, enterovirus, and several other RNA and DNA viruses and diseases that persist within human societies. Bioinformatics paves a clear path; a narrative to how these viruses and diseases carry out their protein expression, and which ultimately lead to a modification of human host cells upon infection. We can gain much knowledge from these expression profiles. By applying established and novel biological methodologies and analytical techniques, a strong foundational base can essentially create a ‘backbone’ of data which can be fit to bioinformational algorithms, and concurrently to statistical packages. What this then does, is provide deep insights and complementary datasets into certain aspects of human diseases; for example: • Which protein(s) are responsible for pathways that lead to expression of host cell receptors that elicit T-cell helper and B-cell responses? • Are there relationships, or lack of, which can be determined from transcriptional abundances gained from experimental studies, which are drawn from the application of informatics programs? • What predictions can be made, in terms of describing epitopes along peptides which combine with MHC Class I and Class II molecules? Can these predictions be used to create vaccine outcomes? • Are there multiple-epitopes that can be uncovered in other variants of the same disease class? This can further provide a more robust vaccine-prediction model based on a combination of immune-informatics and experimental studies. • From the gathered datasets, can a whole-cell focus be concluded? Understanding under what conditions, which ‘pathways’ from host cell contact to symptoms are up-regulated or more frequently encountered. These are just some of the associated questions which can be probed for further investigation when considering a combinatorial approach of ‘wet’ techniques and bioinformational analysis into, for example, endemic viruses and their end-product infection in humans. The challenge in biomedical science now lies in developing stronger algorithms and prediction models which can be readily and appropriately used to derive information which can lead to the synthesis of synthetic vaccines or vaccine adjuvants. Without doubt, a classic example is the lack of knowledge in multiple-epitope peptide-based vaccines; which can be significantly investigated by applying immuno-informatics and immunoproteomics methodologies. These emerging trends in immune-based In Silico approaches will be the next steps in uncovering more accurate information which will not only target more sensitively peptides involved in human virus infections; 1! of ! 19 but which will also enable a new line of peptide-based treatments which can be targeted to specific variants of certain viruses and diseases. So far, peptide-based, multi-epitope, vaccines are a growing market. Their research and progression into immunology will bring to light the many advantages and human health outcomes which currently await our discovery. There are other areas which can be embedded within the ongoing, contemporary, holistic layering within biomedical science research. These include 3D cell culturing; where cells can be harvested and their cellular contents analysis based on their interactions within a 3D environment. Lactic Acid bacteria (LAB) are also an emerging tool. In this approach, a ‘signal’ and ‘anchoring system’ are implemented to display proteins, peptides; consisting of antibodies and/or antigens. The benefits of applying LAB surface display systems in conjunction with bioinformatics systems, is that isolated MHC and peptides can be expressed and displayed on LAB which can then ‘mimic’ or ‘reenact’ how these MHC ClassI/II—peptides elicit a specific T- and B-cell reaction in vitro. Another area of interest could be the application of CRISPR/Cas9 in developing a suitable approach at creating The result: epitopes along the MHC complexed with peptides can be more precisely determined; this can be used to create synthetic peptide vaccines. These are just some of the advantages of combining a multidisciplinary, holistic methodology into human disease and virus research. 2.0 APPROACH TO RESEARCH Item No. Description Method 1 Understanding what vaccines are currently in the literature; this includes sifting through online journals and individual, stand-alone research which describes where dengue vaccine research is currently positioned. Basic investigation and inquiry into the literature. 2 Applying a multi-epitope design to strengthen the immune response upon infection with Dengue virus Using a schematic work-flow based on an immuno-informatics approach. 3 Developing a protocol that details theory into investigating the MHC-peptide complexes and the associated epitopes across said peptides. 3D cell culturing; cell behaviour/expression patterns. LAB surface display of MHC-peptide complex; NIsin Controlled gene Expression system (NICE). CRISPR/Cas9 system. Immuno-proteomics studies. 2! of ! 19 This approach is to gain a deeper understanding in dengue. The focus is not only towards how the virus infects human host cells, but also based on the immunological response; based upon MHC-I/II and T-cell surveillance and effect. 4 Applying informational prediction programs and Immuno-informatics; In Silico approaches. approaches which map peptide epitopes for early stages of synthetic vaccine development. 5 Enlisting appropriate assaying methods to T-cell and antibody assays; ELISA. accurately mine for T-cell responses. These must be affiliated with antibody responses (B-cell) which can then be experimentally tested against, and confirmed, Dengue serotypes. 6 Development of a range of Dengue synthetic peptides; based on combined proteomics, bioinformatics, and assaying methodologies. Combination of applied techniques. 3.0 METHODOLOGY Work Flow Step Description 1 Obtain Dengue serotypes; DENV1-4; culturing of serotypes. 2 Using immuno-proteomics to isolate and fragment peptides associated with MHC Class 1/II. 3 Sequencing of isolated peptides 4 From the Dengue serotypes, classify those peptides that show conserved epitopes and/or similar sequence information. 5 Screen peptides against T-cells (T-Cell epitopes) 6 Apply bioinformatics to investigate a multi-epitope peptide which can yield responses across Dengue serotypes. 7 Carry out in vitro (and also 3D cell culturing) systems to elicit a T-cell and associated B-cell response. i.e. antibody response to antigenic peptide(s). 8 Developing a CRISPR/Cas9 system which can be experimented in Dengue virus and associated immune responses in vivo. The in vivo aspect can be associated with Step 7 (3D Cell culturing) 9 Cloning of selected peptides showing multi-epitope characteristics, and an anchoring/linker system into LAB cells and monitoring this process. The results are to confer membrane-bound expression/display. 10 Peptide candidates; to create synthetic peptide-based vaccines based on a broad range of interaction of peptides to HLA types. 3! of ! 19 3.1 Work flow breakdown 1. Obtaining Dengue virus serotypes is the first step in the process of mapping peptide epitopes. Having a stock of serotypes (1-4) enables a more comprehensive coverage of analysing MHC class I and II along with their associated peptides; and subsequent epitopes. 2. Culturing dengue serotypes in 2D and 3D cell culturing environments. The addition of a third dimension will allow for an additional layer of knowledge, if present, within dengue expression patterns during host cell invasion. These studies can also lead to whole-cell analyses which provide detailed data on metabolites and protein expression abundances; to be used in informational modelling. 3. Applying immuno-proteomics, chromatographic, and also proteomics techniques to isolate unknown peptides bound to MHC Class I and II carriers. Isolating these unknown peptides can also be accompanied with previous culturing outcomes; i.e. tracking whole cell behaviour the lead to peptide-MHC (pMHC) molecules. 4. Sequencing isolated peptides. This must be analysed in conjunction with triggered T-cell responses; measured via an appropriate assay. The assay can be a sub-sample taken during culturing step(s). Sequencing will be based on both the MHC-peptides under investigation, and also the T-cell receptor sequence. Peptide sequences can then be traced for their epitopes (T-cell epitopes), and B-cell activation. 5. Using a bioinformatic pipeline to annotate all sequenced MHC bound peptides. Once this step has uncovered peptide epitopes responsible for binding T-cell receptors, then peptide sequences should also be mapped to rule out any homology within the human genome. 6. Analysing peptides that occur across all four dengue serotypes. The goal is to uncover those peptides which share epitope similarity/homology across all Dengue serotypes. This can rule in a more precise Tcell response that can show later efficacy against all serotypes. 7. Synthesis of selected multi-epitope peptides. 8. Testing synthetically produced peptides within a controlled assay. Assay must induce T-cell response and must be followed with a suitable bioinformatics pipeline. 9. Sequencing of synthetic peptides along with T-cell receptors. This step will ensure peptide epitope interactions match with naturally occurring MHC-peptide and T-cell response in earlier step(s). 10. Re-screen peptide candidates; in vivo models and 3D cell culturing applying suitable scaffolds and matrices. 4! of ! 19 4.0 EXPERIMENTAL DESIGN 4.1 Multi-epitope design: Immunoproteomics and Immunoinformatics Multi-epitope vaccines (MEV) consist mainly of CD4+ and CD8+ epitopes. Broadly, there are two types of epitope-based vaccine which can be employed to combat human disease and viruses; these include: • Minimal-length epitope peptides: poorer immunogenicity. • Longer length (multi-epitope) peptides: eliciting/inducing a strengthened and specific immune response. These include B-cell and T-cell epitopes. They are a safe, stable, cost-effective, and highly specific option. As a multi-epitope vaccine requires the establishment of an immunoinformatics approach, the following guidelines will enable this to be achieved: 1. A selection of peptide antigens. This step should be aligned with concurrent proteomics and bioinformatic studies. 2. Epitope identification; epitope-based vaccines can be either minimal-length epitopes or longer peptides made up of a series of epitopes. This triggers a B- and T-cell response; i.e. humoral and cellular response. 3. Structural analysis of multi-epitopes and MHC Class (I/II) molecules; the arrangement of these epitopes (linear and/or branched). 4. Physicochemical evaluation of peptide sequence and epitopes; solubility, molecular weight, half-life, and pI. 5. Molecular dynamics (MD). This step can be utilised to study biological molecules; by studying interactions and stability of peptide vaccines over a time-frame. The relevant computational portion of identifying and characterising peptide candidates for use as peptide vaccines in dengue is based upon the application of immunoinformatics. In this approach, an ‘immunomic’ map essentially details the immune reactions that occur from a host cell interacting with a foreign particle. This is further complemented with In Silico methods, which can be used to better predict several criteria of a given epitope. this is not a standalone feature of research into developing a peptide vaccine against dengue; and, immunoinformatics is an additional layer that supports immuno-proteomics and in vitro experiments which supports a more robust and confident outcome when selecting and synthesising candidate peptides for novel vaccine formation. Overall, the goal of applying a bioinformatics pipeline to uncovering multiple-epitopes across dengue MHC-peptide display is to observe the impacts of these upon T-cell immunity. That is, to better understand which epitopes are presented across dengue serotypes. To create a universally-relevant Dengue vaccine that is effective across a range of populations, it is worth considering the appropriate activation of various HLA T-cells elicited by peptide epitopes. The vaccine formulation should contain peptides which can bind varying HLA alleles. In Dengue, a strong and broad CD8+ T cell response can ensure a lasting protection. By including a immune-proteomics design, several peptides linked to MHC molecules can be mapped and classified, according to MHC classification, and subsequently 5! of ! 19 investigated for multiple-epitopes. As mentioned, the benefits of this approach lie in designing synthetic peptides which can induce a strengthened T-cell response across all four Dengue serotypes. What is also necessary when enlisting an immunoproteomic approach to find multiple epitopes across Dengueinfected cells, is to describe which naturally presented epitopes are linked to the 10 proteins that comprise Dengue virus; i.e. capsid (C), envelope (E), prM (M), and other NS proteins (Figure 1). Understanding which of the Dengue proteins are expressed, provides a greater insight into those proteins being incorporated as T-cell epitopes. One terrific advantage of utilising an immune-proteomics approach in discovering multiple-epitopes is the the system is unbiased; since the presented peptides conjugated to MHC molecules represent the naturally occurring mode of antigen presentation. This is a crucial element in subsequent peptide fragmentation. This should not rule out the potential of algorithm prediction models; i.e. immuno-informatics, which specify binding scores to a range of HLA alleles. As prediction models do not consider this natural state of MHCpeptide epitope display; they do provide a secondary study which can be juxtaposed to proteomic studies. This can yield the following: • Combined proteomics and immuno-informatics that can reveal similar or closely related peptides from both in In Silico and experimental study. Currently, In silico and associated informatics are still in an infant stage of development, and therefore must be investigated when dealing with epitope-based vaccines. • Peptides found in their naturally expressed form, (MHC-peptide) from proteomics analysis, can be optimised via the use of an immune-informatics work-flow. Nonetheless, incorporating both a proteomics and informatics design, can greatly assist in mining for multipleepitope peptides which can then lead to categorising a stringently selected list of epitopes that appear across multiple populations of Dengue-infected cells. This will greatly improve peptide processing for subsequent display and effect of T-cells. This can be readily stored within an appropriate database; where this information can serve as the basis for further studies; for example in utilising CRISPR/Cas9 and lactic acid bacteria, the following steps that occur in this research report. Lastly, the difficulty will be in the design formulation and mode of delivery of such multiple-epitope peptide-based vaccine. This needs to be considered, as stimulating a CD8+ T-cell immunity will require a delivery method where peptide formulations can survive in vivo long enough to elicit their intended effects. Figure 1: Images taken from Perera et al., 2008. Peptides with epitopes relevant to eliciting a strong Tcell response can be derived from any of the structural and non-structural proteins (above) which form the entire ssRNA dengue virus (right). 6! of ! 19 4.2 CRISPR/cas9: Components and incorporation into a LAB system. Bacteria and archea carry out their natural immunity via the use of the CRISPR/cas9 system, which prevents infection from bacteriophages. Its origins are in bacteria; in which the CRISPR/Cas 9 system is part of a defence mechanism to ward off foreign elements, such as bacteriophages. In short, it is an evolutionary defence against non-self elements. It can also be used as a tool for genome editing and engineering which can be readily implemented within biomedicine. Briefly, it works via an RNA-programmable molecular scissors which cut away at specific genes; which can then manipulate genes and their expression in cell types and organisms. The CRISPR/Cas9 system is comprised of RNA components and protein components which work in unison in ribonucleoprotein complexes which interfere with invading nucleic acids. Breaking down the RNA components, there are one or two types; CRISPR RNA’s (crRNAs) and a second type, entitled tracrRNA which is associated with the type II system. The mode that the CRISPR/Cas9 system carries out its effects upon foreign invasion is based on two events: 1. Memorisation of a genetic element upon a first infection; this is referred to as adaption. 2. Destruction of the same element upon a second infection; known as interference. Adaption is classified as a process which involves Cas proteins recognising an invading mobile genetic element. These Cas proteins insert a short sequence of the invading element into a CRISPR array, which is located along the bacterial genome. This leaves the host with a ‘genetic memory’ which reinvents the host from being reinfected by the same element. The produced library go on to be transcribed via processing events which end in the generation of short mature crRNAs; each of these containing a unique sequence from the memorised genetic elements encountered in the first steps of recognition. Interference is what the name suggests. The same crRNAs are incorporated into Cas proteins into a CRISPR ribonucleoprotein complex (crRNPs) which guide sequence-specific degradation of foreign DNA or RNA. These events describe DNA targeting, however Dengue is a single-stranded RNA virus. What should also be considered in this respect, is that RNA targets based on the Cas9 genes have yet to be explored in Dengue vaccine work. In this instance, utilising the FnCas9 as a programmable RNA targeting strategy; which can further help understand how these can proteins interact with Dengue virus transcripts in vitro. (Figure 2). This essentially renders the FnCas9 molecule as a form of RNA interference, which can then lead to degradation products, for example in the host cell, that can go on as displayed peptide epitopes. FnCas9 can be engineered and also exploited to a RNA-directed RNA targeting system, which can be applied in the scope of developing multi-epitope peptides in Dengue. This can be achieved via the use of plasmid expression systems, expressing specific Dengue structural and/or non-structural genes that can also be co-cultured with other cloned plasmids carrying genes pertinent to LAB-anchroing systems. 7! of ! 19 Figure 2: CRISPR/Cas9 mechanism; based on FnCas9 interaction with an RNA target. (A) In this example, FnCas9 forms a dsRNA complex with two RNAs; tracrRNA and scaRNA. Combined, this complex allows the tracrRNA to track and target an mRNA transcript. This then leads to degradation; the events and mechanisms for this degradation are still being investigated. (B) This depicts a reprogrammed tracrRNA:scaRNA hybrid, which can then target an incoming mRNA. The FnCas9 system of targeting RNA instead of DNA, can be utilised in the Dengue virus; as it is a ssRNA molecule. Further information can be derived, for example by elucidating the effects of FnCas9 in breaking down Envelope and/or prM mRNA transcripts. 4.3 Applying Lactic acid bacteria (LAB) in peptide/MHC surface display. Lactic Acid bacteria (LAB) are a group of gram-positive bacteria which produce, during fermentation, lactic acid. LAB can survive the acidic environment of the gastrointestinal tract (GIT). L. lactis are also nonpathogenic, non-invasive, and make good candidates as live vehicles; to deliver heterologous proteins involved in vaccine research. Lactococus lactis which represent the most commonly sued LAB in protein expression, survive only for a limited time in the GIT. Additionally, the World Health Organisation (WHO) has deemed LAB to contain health-promoting properties; and their resilience in the GIT make them excellent candidates which can be used to deliver vaccines. Applications of LAB can include displaying antigens, antibodies, enzymatic display on LAB surface, and also investigating adhesion as to provide a heightened immune response. Of particular interest, in this study, is the incorporation of LAB systems in antigen representation. The process, however, is not simple, and in order to display a protein or peptide on the LAB surface several components must be considered; signal peptide and an anchoring domain. The further biding of the candidate peptide(s) can be achieved using transmembrane anchors, lipoprotein anchors, and/or a peptidoglycan anchor. Since peptide vaccines require an immunogenic peptide to elicit an immune response, in dengue. The application of LAB, along with the testing and subsequent optimisation of necessary anchors and signal sequences would have tremendous potential when designing and testing efficacy in novel dengue derived peptide-MHC complexes, post peptide cleavage using immunoproteomics. What can also be carried out using Lab systems is the anchoring of peptide-MHC-I/II complexes to LAB membrane surfaces; this can provide additional information regarding which peptides epitopes are responsible in T-cell responses. Finally, the use of LAB as presenting peptide-epitope vaccines is a safe way to carry out later studies in human subjects, if results permit validity. 8! of ! 19 Figure 3. What is of attractiveness is the ability to use LAB as delivery vehicles to express protein/ peptide antigens with multiple epitopes. This could be a promising synthetic peptide used to elicit a Tcell response, or a series of peptides based on the immunological responses from a combined effect of several Dengue virus processed proteins, traditionally displayed within MHC molecules. Image taken from Wang et al., 2016. Figure 4. An overview of the different membrane anchoring methods in lactobacilli. Each example represents either a covalent or non-covalent anchoring system. Using a combination, or standalone anchoring system, Dengue virus processed peptides, such as those belonging to Envelope or prM proteins can be effectively displayed based on their immunoinformational multi-epitope discovery. These studies can lead to developing a Dengue vaccine which can be readily consumed by humans. Image taken from Michon et al., 2016. 4.3.1 Using the Nisin-Controlled gene Expression System (NICE) The NISIN-controlled gene expression system (NICE system) is a gene expression system based on the auto regulation mechanism of nisin biosynthesis in L. lactis. It works by controlling gene expression, in which nisin serves as an inducer. The PTM antimicrobial peptide nisin, functions as a signal peptide, inducing the transcription of the nisin gene cluster; nisABTCIPRKFEG (Figure 6). Nisin functions in the following manner: firstly, it binds to the N-terminal domain of NisK, and NisK autophosphorylates transferring a phosphorous group to intracellular NisR. This then goes on to as a transcription activator of nisA/nisF, to subsequently induce gene expression. Figure 5. NICE System Nisin (red swirly line), an antimicrobial peptide, induces expression of the nisin gene cluster. Once nisin induces a two-component regulatory system; which consists of 1) histidine protein kinase (NisK), and, 2) the response regulator (NisR). Image taken from Zhou et al., 2006. A series of lactic acid strains and plasmids can be utilised to express nisin, which can then function as to express membrane surface genes of interest, within appropriate vectors, which can then elicit a desirable effect. Additionally, nisin is already commercially used within the food industry; meaning, nisin is a food-grade inducer whose functions can be directly transferred into human host vaccine research. Nisin can essentially be used to express lethal genes, such as those associated with Dengue; i.e. prM (M), and envelope (E) proteins. 9! of ! 19 Nisin ensures a tightly regulated expression in an uninduced state, and this is of particular interest when developing a multi-epitope peptide which derives its sequence from fragmented PrM and/or E proteins. Oevrexpression of these Dengue proteins can also be achieved using the NICE system. In this scenario, and depending on the presence or absence of a signal peptide such as the Usp45, the desired protein can be expressed in the cytoplasm, membrane, or into the surrounding medium, which can then be targeted via the action of FnCas9 (Section 4.2) to produce a memory of infection (adaption) from the necessary RNA degradation products (interference). 4.4 3D Cell Culturing It is important to consider the spatial environment when dealing with specific cells to be investigated in virus research. One attractive method is to recreate a growth environment which mimics the native tissue environment, and allows for cells to proliferate around and within a scaffold structure. By producing a cellular environment that is similar to in vivo conditions, models for drug screening can be developed. Along with this, applying 3D cell culturing systems can reduce the gap between these culturing systems and cellular physiology, particularly in disease/virus research. The drawbacks of 2D cell culture methods is that much data relating to cell-to-cell interactions, and also cell proliferation is lost or not taken into account. Other benefits include a more cost-effective screening platform for developing novel drugs; this includes peptide-based vaccines in dengue, and also provide higher value when undergoing later safety and risk assessment. 3D cell culturing can be investigated in this study; specifically by allowing host cells and dengue virus particles to undergo contact and further proliferation. Once the RNA replication has been fully achieved, gene and protein expression profiles based upon peptide-MHC complexes are just one feature which data can be retrieved and further scrutinised using bioinformational software to characterise how these peptides-MHC and T-cells influence the environment around them; i.e. in a near-to-in vivo culturing system. 3D cell culturing systems also have the potential to validate peptides fractionated from chromatographic techniques followed by mass spec analysis. This provides an in vivo validation, and supplementation, which can be used in conjunction with animal models. What is now required is a sort of ‘virus on a chip’ approach; which can provide a mimicking of human physiology on a micro-scale. Dengue is no exception to this approach, and careful consideration should be given to applying a 3D cell culturing system to this virus, as animal models can lead to toxicity during initial trials. It seems that translating information from 3D cultures into human vaccine outcomes is a very likely possibility in the field of cell culturing. As vaccine development has relied on animal models to probe into the types of immunological responses; now, 3D cell culturing can be applied in virology and vaccinology to assist in multi-epitope peptides which can then carry on to a bioinformational stage. One last goal which can be achieved, over time, is the ability to develop a series of ‘virus on a chip’ products which can provide researchers with a realistic ‘overview’ of an entire virus operation, given a very specific environmental scenario; i.e. differing serotypes of Dengue which exist in global Dengue virus hotspots. This can be applied to Dengue studies; which can provide an over-arching ‘omic’ readout of all activities taking place in human host cell infections. The analysis can be complemented with bioinformatics. ! of 19 10 ! 4.4.1 Training bioinformatics programs from 3D culturing datasets Since the concept of providing an additional layer of cellular behaviour from 3D culturing assays has become confidently documented in an Alzheimer's Disease study more recently (Kim et al., 2015), its advantages can be linked to serve as a ‘training set’ for further bioinformatics platforms. Figure 5 outlines and describes the inclusion of training data onto bioinformatics platforms, in a diagrammatic format. Studying Dengueinfected host cells combined with an appropriate scaffold/ matrix to study cell behaviour. Scaffolds/matrices: (thick black lines) - Biological and synthetic hybrids. - ECM - Collagen Attributes: Cell signalling Gene expression Physiological triggers Transcription factors Metabolite profiles Biomarkers Figure 6. (Above): A specific scaffold/matrix should be associated with the cell type and study in question. (Right): Step-wise process of subsequent ‘dataset training’ approaches. Training 3D Dengue cultured cells onto software programs will provide near to real conditions which can substitute animal studies. 1) Train bioinformational programs on derived datasets. 2) Use developed and existing prediction models to ascertain multiepitope peptides displayed on MHC Class molecules, based on trained data. 3) Align concurrent/previous immunoproteomic studies with prediction outcomes; focusing on peptide-MHC complexes. 4) Carry out further assaying to elicit immunological responses from selected multi-epitope peptides. 5) Select screened multi-epitope peptides In this regard, this research study will apply 3D culturing results that can be readily implemented to informatics platforms that can provide a more realistic and ‘in-vivo’ remodelling of Dengue. 4.5 Creating a Promising Dengue vaccine combining all approaches What is described above, can be summarised as the following: • Designing an experimental study which can incorporate emerging, Cas9, discoveries in tropical disease research. • Moving towards a more closely-related, animal-free, testing platform which encompasses computational and molecular biological techniques to gain a deeper understanding of how Dengue produces MHC and peptide molecules. • Targeting peptides which are found to contain multiple sites of strong T-cell triggering epitopes which an be purified, and expressed in suitable vehicles; for example lactic acid bacteria, along with the associated cloning of these peptide fragments and their epitopes, and membrane anchoring proteins which can effectively secure these antigens. ! of !19 11 • Using appropriate assaying in conjunction with LAB membrane protein expression systems (covalent and/or non-covalent) to uncover T-cell and B-cell immunity when cultured with Dengue (serotype) infected host cells. This system can also test for the anchoring MHC molecules across the membrane of LAB; in effect, a MHC-peptide-anchoring conjugation. • Screening and re-screening strong multi-epitope elicitors which can be refined and published for further trials. ! of 19 12 ! 5.0 JUSTIFICATION OF STUDY Informatics, multi-epitope candidate studies, and emerging technologies combined, are necessary platforms that will link and narrate the dengue virus research to validate data which can direct research efforts to a peptidebased vaccine outcome. The result: defining, and optimising a multi-epitope peptide-based vaccine which can be readily administered via LAB, for example. Currently, there has been little progress made to develop a multiepitope vaccine for dengue virus serotypes. By opening up research into investigating dengue serotypes and the synthesis of a novel peptide vaccine which can combat all four serotypes a greater understanding of which peptide-MHC Class I/II complexes, can be elucidated as repertoires of peptides can be screened for their potential as therapeutic candidates. Aligned to this approach, the current advances in technology, such as the above mentioned in 3D cell culturing, CRISPR/Cas9, bioinformatics, proteomics, and lactic acid bacteria are all conducive to obtaining results that reinforce an effective strategy to finding a vaccine against dengue. These approaches, as a collective, have yet to be investigated and explored; and this study will provide the foundations and appropriate justification to undertake research activities which can not only answer questions relating to all approaches, combined, but also establish and steer Dengue virus vaccine research into a brand new direction. Broadly speaking, the justification of this study lies in the demand for a dengue vaccine which can address all four serotypes, efficaciously, whilst at the same providing strong science which can be directly translational to support industry and the R&D sector in dengue virus research. As any vaccine development can be a difficult challenge, the approach taken in this study will be one of generating a greater depth of knowledge in regards to implementing recent developments in scientific research, such as immunoproteomics, which will shift dengue research from lab bench to market using a multifaceted approach that aims to more rationally design novel peptides, and their corresponding epitopes as vaccine candidates. What is of equal importance, when justifying this research is industry support and understanding their perspectives when considering investment; if IP, licensing of technology, and patents are to be reviewed as options. Critical questions and inquiries asked from potential stakeholders and industry might include: - What is the end use of this IP (in this case the final Dengue multi-epitope vaccine) and how will it deliver its - mode of effectiveness in humans? Which competitors are addressing dengue virus? What is the competitive advantage of this IP? How does this vaccine deliver optimal results when compared with current Dengue vaccines? Is the designed vaccine deemed safe and consumable by humans? Is the pipeline/timeframe feasible? That is to be considered to bring this vaccine to market, in a timely manner? These questions can be answered using a project management approach which can provide stakeholders and industry professionals with adequate and appropriate responses which can attract further support and investment. ! of 19 13 ! Of course, the lengthy time of vaccine development to human-health outcomes means that this multi-epitope vaccine can be optimised; with time available for publications and industry liaising. In light of this, it is also critical to seek out, and consider, commercialisation branches that can offer detailed assessment of the developed IP. So far, there currently exists six dengue virus vaccine candidates. These are listed below in Table 1. These vaccines do not include peptide-based vaccines; as either a primary constituent or adjuvant in their formulations. This then provides further justification to explore the potential efficacy and effectiveness of multi-epitope peptides as vaccine candidates. Table 1: Dengue virus vaccines currently in clinical trialling. Vaccine Name Current stage of clinical trials Mode of action/description CYD vaccine Past Phase III: Registration (Sanofi Pasteur) Live attenuated vaccine that uses the yellow fever virus as a suitable backbone; prM and E proteins from a wild type dengue virus have been substituted into the yellow fever vaccine. DENVax Phase I Mix of live-attenuated DENV2 and chimeric DENV1, DENV3, DENV4, which is based on the attenuated DENV2 backbone. Strains for serotypes 1,3,4 were made by replacing the prM and E proteins of the DEN-2 PDK53-V, with genes from wild-type serotypes. TV003/TV005 Phase II (NIAD) Phase III (Butantan Institute) Phase I (Panacea Biotec) Full-length DENV1, 3, 4 and a chimeric DENV-2 vaccine; based on a wild type DENV1 and DENV4 strain with a 30 nucleotide deletion in the 3’-UTR. V180 Phase I (Merck) Recombinant subunit based on the dengue wild type prM and a truncated E protein through expression in a Drosophila S2 cell expression system. TDENV PIV1 (TDENV-LAV + TDEN-PIV2) 1 Phase I (GSK) 2 Phase I (WRAIR) Tetravalent purified inactivated vaccine; also being tested with other adjuvants. D1ME100 Phase I (Naval Medical Research Centre) Monovalent plasmid DNA vaccine; prM and E proteins of DENV1 expressed under the control of human cytomegalovirus promoter/enhancer of plasmid vector: VR1012. Vaccines have included those affiliated with LAV, using mainly Dengue virus structural proteins in their development. This study aims to produce data pertaining to creating confidence in the search for multi-epitope peptide candidates which can be subsequently transferred to novel LAB delivery systems. What has yet to be investigated is the incorporation a CRISPR/Cas9 approach in Dengue research. As CRISPR/ Cas9 (FnCas9 for RNA) is an innate immune defence in bacteria, LAB can be a suitable candidate that can ! of 19 14 ! establish the role of CRISPR/Cas9 and also its effects of interrupting foreign nucleic acids, such as Dengue virus (ssRNA) and its associated structural and non-structural proteins. No data is currently available on from the combination of LAB, CRISPR/Cas9, and appropriate in vivo-like cell culturing in Dengue. 6.0 MOTIVATIONS OF STUDY AND DELIVERABLES With a greater understanding of bioinformatics and techniques such as CRISPR/Cas9 and the more accepted role of LAB, along with more precise instrumentation that can analyse samples containing antigenic peptides bound to MHC-I/II; there is now greater space to fill with vaccine innovation. Little is known regarding the mechanisms of utilising a lactic acid bacterium along with, and within, a CRISPR/Cas9 system to study Dengue virus behaviour in vitro; and this study will be the first to include a holistic research approach in these areas. As of 2017, there has been only a handful of studies that have looked deeper into multi-epitope peptides that can be utilised in a stand-alone or adjuvant vaccine formulation. This leaves a wide knowledge gap that should be addressed with a collaborative methodology. This approach will include: 1. Bioinformatics and derivative proteomics to assess epitopes presented on MHC peptide complexes. 2. A 3D cell culturing system, within a bioreactor system for example, which provides greater depth to study cell physiology of LAB when expressing foreign proteins; those associated with Dengue, i.e. E and M proteins. 3. Cloning CRISPR/Cas9 components, along with NICE-system elements, and multi-epitope peptides (from point 1) into a whole-cell (LAB) system, and tracking the events of this conjugated expression. By applying the above, designed to produce a new model of the dengue virus, and by strategically utilising current next-generation analytical techniques, outcomes in a multi-epitope peptide vaccine can be developed. Additionally, appropriate immunological responses, a shortened time-to-market vaccine, and greater experimental potential with statistically significant results, can be guided from computational predictions and experimentally studied epitope and contemporary analytical techniques. Deliverables and outcomes are listed below; and, are brief in their description. 6.1 Research-based: • Accessing computational programs that aid in optimising the uncovering of dengue virus multi-epitope peptide-MHC complexes. This approach is specific and focuses on applying derived data to dengue virus modelling for epitope prediction based on a stringent criterion. ! of 19 15 ! • Applying immunoproteomic experimentation to characterise peptides purified and isolated from MHC complexes. As a complement to the above point, immune-proteomics can provide the experimental layer which can describe, within an in vivo context, those multi-epitopes involved in producing an appropriate Tcell and subsequent B-cell response. • Mapping, through whole-cell analyses, relevant pathways that correspond to the production of antigenic peptide epitopes and MHC molecule complexes. This information can be further used to address how and which mechanisms peptide-MHC-I/II complexes employ, along with their corresponding T-cell responses. These studies can be supported via 3D cell culturing and LAB experiments, and be used for training informatics platforms. • Applying a CRISPR/Ca9 system to study which Dengue (serotypes) proteins can provide defence during infectivity; preferably in a LAB vehicle. As this experimental step will initially be a stand-alone, the goal will be to further investigate how Dengue proteins can be cloned into selected LAB species, and if a specific CRISPR/Cas9 response can lead to subsequent interference of Dengue proteins, which can then be potentially associated with cloned MHC molecules via plasmid constructs. As this work is relatively new in affiliating CRISPR-Cas9 with Dengue vaccine research; there is data that can be obtained from this combinatory system. • Designing a series of synthetic multi-epitope peptides from which have been screened using computational and experimental analyses. These synthetic peptides can then be tested in standard and advanced assaying parameters for a specific immunoglobulin response that shows promise at neutralising or eradicating dengue serotypes. 6.2 Administrative-based: • Patenting novel sequences, as multi-epitope peptide-based vaccine developments, targeted towards dengue serotypes, or a single serotype. As a lengthy process must be considered when filing for patents, this endeavour is best pursued as a group/team milestone. • Opportunities to partner within the biotechnology start-up space, which can lead to further funding support, and joint-project opportunities. • Clinical trial and ethical consent considerations from peptide-based vaccine. If in vitro studies show a promising CD8+ T-Cell response, suitable ethical considerations can be further explored. • Licensing patented IP to research institutes and organisations that seek to further investigate and use developed peptide-vaccine as a whole vaccine or adjuvant in their dengue-vaccine formulations. ! of 19 16 ! • Strong peer-review articles that confirm and support experimental parameters. As CRISPR/Cas9 has received little attention in terms of Dengue virus research; this research will provide new knowledge which can build upon further publications and expand CRISPR/Cas9 to tropical disease research. ! of 19 17 ! BIBLIOGRAPHY Andrew J. Bordner, 2013. Structure-Based Prediction of Major Histocompatability Complex (MHC) Epitopes. Methods and Protocols, Springer Protocols. Emmanuelle Charpentier, 2015. CRISPR-Cas9: how research on a bacterial RNA-guided mechanism opened new perspectives in biotechnology and biomedicine. 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