In Silico Design and Analysis of an Epitope-Based Vaccine Targeting the VP24 Protein of Ebola Virus for Enhanced Immunogenic Response
Abstract
Ebola virus (EBOV), a highly lethal pathogen causing Ebola virus disease (EVD), poses significant global health challenges due to its high mortality rate and lack of licensed vaccines. This study focuses on the in-silico design of an epitope-based vaccine targeting the VP24 protein of EBOV, a key player in viral replication and immune evasion. The VP24 protein sequence was retrieved from the NCBI database and analyzed using various bioinformatics tools. B-cell and T-cell epitopes were predicted using IEDB and NetMHCpan 4.1, respectively. The selected epitopes were assembled into a multi-epitope vaccine construct with appropriate adjuvants and linkers. The vaccine's antigenicity, allergenicity, and toxicity were evaluated using VaxiJen, AllerTOP v2.0, and ToxinPred. The secondary and tertiary structures of the vaccine were predicted using PSIPRED and trRosetta, followed by structural refinement and validation through GalaxyRefine and Ramachandran plot analysis. Molecular dynamics simulations using iMODS and immune simulations with the C-ImmSim server confirmed the vaccine's stability and predicted a robust immune response. The in silico designed vaccine exhibited high antigenicity, low allergenicity, and structural stability, indicating its potential efficacy. This study demonstrates the feasibility of using computational approaches for rapid and cost-effective vaccine design, offering a promising strategy for combating Ebola virus outbreaks. Further experimental validation is required to confirm these findings and advance the vaccine candidate towards clinical development.
CHAPTER 1
1. INTRODUCTION
West and equatorial Africa are home to endemic Ebolaviruses, which are negative stranded RNA viruses that are members of the Filoviridae family. These public health diseases, which cause severe and acute systemic disease with a high fatality rate, are mainly spread by human-to-human contact with infected bodily fluids and corpses. The West African outbreak of Ebolaviruses in 2013–16 demonstrated the virus's significant potential for epidemics (Lo, Marston et al. 2017). With almost 28,000 confirmed cases and 11,000 fatalities, this outbreak was of a historic magnitude(Garske, Cori et al. 2017). It had a devastating effect on the west African economy. This outbreak also demonstrated how quickly a disease may spread from rural communities that are most impacted to the metropolitan districts of larger cities when public infrastructure is lacking in resources. The outbreak was eventually contained with the help of international assistance and significant efforts from the impacted nations. Another distinctive feature of this outbreak was that it prompted extensive research programs on ebolavirus-related pathology to be started and carried out, which has resulted in significant scientific advancements. Ebolaviruses are viruses having a single, negatively polarized strand of RNA making up their genome. They are classified under the genus Ebolavirus in the family Filoviridae within the order Mononegavirales. The five species that make up the genus Ebolavirus are known by their taxonomic names: Burdibugyo ebolavirus (Bundibugyo virus), Reston ebolavirus (Reston virus), Sudan ebolavirus (Sudan virus), Taï Forest ebolavirus (Taï Forest virus), and Zaire ebolavirus (Ebola virus).
1.1 Historical perspectives
The unexpected development of ebolaviruses in 1976 in remote regions of Sudan (approximately 850 kilometers from the Zaire epidemic) and Zaire (now the Democratic Republic of the Congo) startled medical professionals, garnered international attention, and frightened residents of the epidemic area. identified as a novel viral hemorrhagic fever very away. EVD joined a growing list of other viral hemorrhagic fevers, including the centuries-old yellow fever, the 1944-first-recorded Crimean-Congo hemorrhagic fever, the 1951 Korean hemorrhagic fever, the 1953 dengue hemorrhagic fever, and the 1955–1969 Argentinian, Bolivian, and Lassa (arenavirus) hemorrhagic fevers. Despite their varied origins, modes of transmission, natural histories, and pathogeneses, these agents all share a severe systemic viral infection linked to one or more hemorrhagic phenomena, including frank bleeding, ecchymoses, or petechiae. Investigations revealed that two phylogenetically related but genetically distinct ebolaviruses, the Sudan ebolavirus and the Zaire ebolavirus, were responsible for the epidemics, despite the initial assumption that the two proximate 1976 ebolavirus outbreaks were a single event, in which the arrival of an infected traveler had seeded one or the other area. The most likely explanation, according to virologists and epidemiologists, was that the viruses separately spread into humans after emerging from two distinct reservoirs (for example, two distinct mammals) under the influence of a common cause.This theory was supported by research from numerous other sylvatic viruses, which showed that viral reservoir hosts are usually species to which a virus becomes stable over an extended period of time, causing little to no disease in that host and being sustained by silent circulation within the host species, either through host-to-host transmission or through intermediate hosts or vectors (Kuhn, Adachi et al. 2019). Trapping and bleeding thousands of animals and insects in the years after the 1976 ebolavirus outbreaks did not reveal a reservoir. Later, it was discovered that chimpanzees, gorillas, and other mammals experienced epizootics and ebolavirus emergences, but only as infected dead-end hosts, similar to humans, when the virus emerged from its unidentified reservoir or reservoirs. Fieldwork conducted over the past 30 years has revealed that ebolaviruses can spread throughout sylvatic ecosystems throughout the African tropical belt. This is particularly true for a number of fruit bat species, which have been hypothesized to be either reservoir hosts or somehow involved in the emergence of ebolaviruses. Due to its greater medical and public health significance, as well as the fact that a far greater amount of research has been published on it than on other ebolaviruses, this discussion focuses on EBOV as the species of Zaire ebolavirus. With the exception of members of the species Reston ebolavirus, which appear to be nonpathogenic for humans, evidence does not suggest significant differences in human pathology or pathogenesis between the ebolaviruses. A thorough examination of the filovirus family was just completed.
1.2 Ebola Virus Biology
These viruses are classified by the Baltimore V system as belonging to the family Filoviridae of the order Mononegavirales, which also includes the three genera Ebolavirus, Marburgvirus, and Cuevavirus (Burk, Bollinger et al. 2016). The five viruses that make up the genus Ebolavirus are Sudanebolavirus (SUDV), Reston Ebolavirus (RESTV), Bundibugyo Ebolavirus (BDBV), Taï Forest Ebolavirus (TAFV), also known as the Ebola virus of Cote d'Ivoire, and Ebola virus (EBOV, formerly known as the Zaire ebola virus) (Kuhn, Andersen et al. 2014). The taxonomy of filoviruses has been revised and improved in recent years. To address issues with the 2005–2011 classification, the International Committee on Taxonomy of Viruses Filovirus Study Group recommended a number of modifications to the filovirus taxonomy in 2011. They underlined that phrases that are similar can mean distinct things: While EBOV is a virus that belongs to the genus Ebolavirus and species Zaire ebolavirus, Ebolavirus and Zaire ebolavirus are taxonomic terminology (Bukreyev, Chandran et al. 2014). EBOVs range widely in size, with diameters ranging from 50 to 80 nm and lengths ranging from 10,000 to 14,000 nm. Cylinders, branches, and loops are among the general shapes of virions. Nonetheless, all filoviruses retain their distinctive filamentous structure that resembles a thread (Majid, Tahir et al. 2016). The huge, non-segmented, negative-strand RNA of EBOVs is roughly 19 kb in size. Seven genes are ordered consecutively in this RNA genome. The genes consist of the RNA dependent RNA polymerase (L) – 5’ trailer, the 3′ leader – nucleoprotein (NP) – virion protein (VP) 35, the matrix protein VP40, the glycoprotein (GP), the VP30, the VP24, and the VP35. With the exception of GP, which codes for three glycoproteins, each of these genes produces a single protein product (Volchkov, Volchkova et al. 1999, Yu, Weng et al. 2017). Conserved leader and trailer sequences with replication promoters and RNA packaging signals distinguish these genes. The 3′ and 5′ untranslated regions (UTRs) flank each gene. Conserved transcriptional signals that start toward the 3′ end of the genomic sequence with a start site and finish with a stop site serve as their boundaries. Furthermore, the majority of the genes are separated by intergenic regions of varying length. However, the existence of gene overlaps in portions of their UTRs—that is, the stop site of an upstream 3' gene overlapping with the start of another downstream gene—is a peculiar characteristic shared by all EBOVs (Baseler, Chertow et al. 2017). The genetic diversity among EBOVs species ranges between 25 and 35% (Singh, Dhama et al. 2017). Gire et al. (Gire, Goba et al. 2014) found several mutations that produce genetically distinct sequences, showing that the source of new lineages during outbreaks are from a common ancestor. NP encodes for a structural nucleoprotein, VP40 and VP24 for matrix proteins, and GP for envelope glycoproteins. The main non-structural proteins are RNA polymerase L, VP30, and VP35-encoded polymerase L matrix proteins, in addition to these structural proteins (Singh, Dhama et al. 2017). While each of these proteins has a distinct function, the ribonucleoprotein-RNP complex, which includes NP, VP35, VP30, and L, is necessary for viral transcription and replication. In contrast, the membrane proteins GP, VP40, and VP24 aid in the growth of the filamentous virions.
1.3 Virus epidemiology for Ebola
Since the discovery of ebolaviruses in 1976, more than 20 outbreaks of the disease have been documented in sub-Saharan Africa, primarily in Sudan, Uganda, the Democratic Republic of the Congo, and Gabon. These epidemics are primarily caused by the Ebola and Sudan viruses. The Gulu outbreak in 2000 was in a semi-urban area of Uganda, however the majority of these outbreaks have been in remote rural areas. On the other hand, minor epidemics might not have been recognized as such. The biggest Ebola outbreak to date happened in west Africa between 2013 and 2016, primarily affecting Guinea, Sierra Leone, and Liberia (Briand, Bertherat et al. 2014). It had a relatively high incidence and mortality rate (>28 000 cases with >11 000 fatalities), and it covered both rural and urban regions in several nations. However, the actual load may have been much larger due to underreporting(Garske, Cori et al. 2017). The overall mean case fatality in this epidemic was 62·9% (95% CI 61·9%–64·0%) among confirmed cases with documented clinical outcomes. The most recent outbreaks were reported in August 2018 in the Democratic Republic of the Congo's North Kivu province and in May 2018 in a remote region of the Equateur province (Barry, Ahuka-Mundeke et al. 2018, Mbala-Kingebeni, Villabona-Arenas et al. 2019). When the outbreak reached Mbandaka, a sizable city and a transit hub situated near the Congo River, in May 2018, the situation grew concerning. Thankfully, the outbreak, which resulted in 54 cases overall—33 of which were fatal—was contained somewhat swiftly. The region's ongoing brutal war makes it difficult to contain the outbreak in North Kivu. The Ebola virus is thought to be a zoonotic disease that can occasionally spread to humans, apes, and potentially other animals. Although the Ebola virus has not yet been isolated from bats in their native habitat, fruit bats of the Pteropodidae family are believed to be the virus's natural hosts. Humans are most likely to contract the disease from direct or indirect contact with infected bats or from handling ill or dead infected forest animals (Emanuel, Marzi et al. 2018). Direct contact with an infected person's blood, saliva, or other bodily fluids can result in secondary human-to-human transmission. Nosocomial transmission often happens before the epidemic is detected, which is further explained by the heightened risk of caring for the sick or handling dead bodies (for example, at traditional funerals). There have also been reports of sexual transmission by Ebola virus disease survivors (Diallo, Sissoko et al. 2016). Seroprevalence surveys suggest that asymptomatic infections can occur (Bower and Glynn 2017). Beyond the direct morbidity and mortality attributable to Ebola virus disease, the disease has indirect effects on population health because resources are diverted from programmes aimed at controlling other diseases of major importance—such as HIV infections, (Bekolo, Diallo et al. 2017) malaria,(Aregawi, Smith et al. 2016) tuberculosis,(Ansumana, Keitell et al. 2017) and human African trypanosomiasis—from programmes improving maternal and infant health and from primary care (Sochas, Channon et al. 2017).
1.4 Diagnosis
Although virus isolation in cell culture has been the primary method for detecting filoviruses for many years, real-time RT-PCR testing were the mainstay of the laboratory response during the 2013–16 west African Ebola virus disease outbreak. In the great majority of cases, the viral load is already high and detectable in the patient's blood by RT-PCR when patients with Ebola virus disease arrive at a hospital, usually 3–6 days after the onset of symptoms. (Lanini, Portella et al. 2015) However, if clinical suspicion remains, testing should be done multiple times over a 72-hour period because a single negative RT-PCR result early after onset does not rule out Ebola virus disease(Rieger, Kerber et al. 2016). Three to seven days following the start of symptoms, the viral load peaks. Viralemia is often 10–100 times higher in fatal cases than in survivors. About two to three weeks after the disease beginning, viraemia in survivors will drop to below the RT-PCR detection limit (about 1000 viral RNA copies per milliliter of blood). Since IgG and IgM develop in survivors but not in all fatal cases, serology can only be used to diagnose Ebola virus disease in a small percentage of symptomatic patients. Seroconversion, or a significant increase in antibody titre in paired serum samples, is necessary for an unequivocal diagnosis. To diagnosis paucisymptomatic or asymptomatic Ebola virus infections, which are characterized by very low viraemia and the production of IgG and IgM approximately three weeks after infection, serology is the preferred approach. RT-PCR on an oral swab is the most effective method for diagnosing post-mortem Ebola virus disease, which includes individuals who died in the community without visiting a hospital. Antigen identification by immunohistochemistry on a skin sample is another, but little employed, post-mortem approach (Emond, Evans et al. 1977). Saliva, tears, sweat, breast milk, urine, CSF, ophthalmic fluid, amniotic fluid, vaginal fluid, and seminal fluid are among the numerous body fluids in which viral RNA can be found by RT-PCR throughout the acute stage of the illness and convalescence. After the RT-PCR on blood turns out to be negative, viral RNA can still be found in these fluids. In areas known as immunologically privileged locations (including the eye, central nervous system, and testis) where antiviral immune responses are less effective against the virus, the Ebola virus tends to persist regardless of the severity of the acute illness. Clinical aftereffects, illness reactivation, persistent virus shedding, and virus transmission are all linked to persistence. With a 4-fold decrease in virus RNA concentrations per month, male survivors' seminal fluid can continue to test positive for RT-PCR for months to years after they are released from the Ebola treatment facility (after the acute illness has subsided). For over a year beyond the commencement of the disease, the virus present in the seminal fluid might remain contagious and sexually transmissible (Sissoko, Keïta et al. 2017). Although the period of infectivity through breastfeeding is unknown, cases of women spreading the virus through breastfeeding have been documented. According to reports, there may yet be more human-to-human transmission pathways and reservoirs for the virus that persists in humans (Dokubo, Wendland et al. 2018).
1.5 Prevention and control of EBOV disease
In vulnerable regions with violent wars, food insecurity, and inadequate health infrastructure, scientists and humanitarians are aggressively searching for virus control strategies. Through a multidisciplinary approach that incorporates elements including early case identification, quick isolation of suspected infected patients, proper clinical management, safe burial practices, and health education, disease control aims to stop the virus's transmission and spread (Gray, Stringer et al. 2018). In high-risk locations, surveillance methods should be used to detect cases of EBOV disease early. Specialized labs for quick virus identification and specialized centers for case isolation must be available in every region. During the 2013–2016 outbreak, Ebola Treatment Centers (ETC) were set up in Guinea, Liberia, and Sierra Leone. ETCs are separated into two distinct categories: 1. High-risk locations that house dead bodies, polluted materials, and diseased patients. 2. Low-risk locations featuring nursing stations, offices, storage spaces, and labs. These centers offer high quality care for affected communities through visits to family members to inform them of medical activities and prevention measures. Individual treatment units were created in the DRC to help medical staff be more flexible and accessible in situations where patients did not visit the ETCs. These isolation units are separate, transportable rooms that are close to the patient's house and have a BSL4 degree of containment. The transparent walls make it easier for medical personnel to monitor patients and provide direct communication with the family. One of the most crucial tools for controlling outbreaks is contacting and keeping an eye on the neighbors and family members of affected individuals. Between 2014 and 2015, contact tracking in Liberia enabled the identification of 3.6% of new cases (Swanson, Altare et al. 2018). In addition, traditional burial ceremonies with safety precautions contributed to the effective control of the disease (Tiffany, Dalziel et al. 2017). The community can identify cases of EBOV disease and take necessary action with the support of health campaigns and educational initiatives. Furthermore, these initiatives dismantle cultural norms and ideas that pose a threat to disease prevention. Religious leaders, traditional healers, or those infected with the virus are strongly encouraged to take part in the festivities. Through direct or indirect contact with bodily fluids of infected reservoir animals, infected or deceased individuals, or surfaces and items contaminated with these fluids, humans can contract the virus. The World Health Organization (WHO) and the Pan American Health Organization (PAHO) have developed health prevention programs to stop the spread of disease. These programs include guidelines for cleaning and disinfecting the environment, hand hygiene, using disinfectants to decontaminate surfaces and equipment, using personal protective equipment for caregivers and health professionals, and handling potentially contaminated materials safely (Organization 2014). Additional suggestions for infection prevention and integrated control include the establishment of a committee to oversee operations in communities and hospitals and the safe processing of laboratory samples. In order to build trustworthy relationships with local communities during public health emergencies, effective communication techniques are essential. Biomedical and scientific instruments and expertise must be used to assess the information's reliability and quality (Allgaier and Svalastog 2015). During the Ebola outbreak in West Africa, Claudia Evers, Ebola emergency coordinator at Medecins Sans Frontieres (MSF) in Guinea, said: “MSF made a big mistake. We advocated for an increase in beds for too long, and everyone listened to MSF. Instead of asking for more beds we should have asked for more sensitization activities” (Hussain 2015). The public's hostility against the Ebola response teams was the outcome of this communication breakdown. Both conventional and cutting-edge media, including newspapers, radio, television, and social media, should be used in local and international Ebola communication campaigns. Therefore, for the most effective implementation of the suggested steps, there exist groups like "Translators without Borders" that are professionals with a track record of supporting the communication of public health measures in local languages.
1.6 Objectives of the study
This study aims to design an epitope-based vaccine against the VP24 protein of EBOV using in silico methods. The VP24 protein was chosen due to its crucial role in viral pathogenesis and immune evasion, making it an attractive target for vaccine development. The study involves the retrieval and analysis of the VP24 protein sequence, prediction of B-cell and T-cell epitopes, and the construction of a multi-epitope vaccine. The vaccine's immunogenicity, allergenicity, and physicochemical properties were assessed to ensure its efficacy and safety. Additionally, the structural stability of the vaccine was evaluated through molecular dynamics simulations, and its potential to induce a robust immune response was predicted using immune simulation tools. The outcomes of this research hold significant potential for the development of a novel and effective vaccine against Ebola virus, which could contribute to controlling future outbreaks and reducing the global burden of this devastating disease.
To Be Continue.....