World Journal of Pharmaceutical Research Nandi et al. World Journal of Pharmaceutical SJIF ImpactResearch Factor 8.084 Volume 9, Issue 6, 1265-1287. Review Article ISSN 2277– 7105

RNA GENOME OF VIRION AND VACCINATION BY MONOCLONAL ANTIBODIES

*Kushal Nandi and Dr. Dhrubo Jyoti Sen

Department of Pharmaceutical Chemistry, School of , Techno India University, Salt Lake City, Sector-V, EM-4, Kolkata-700091, West Bengal, India.

ABSTRACT Article Received on 06 April 2020, Ebola, also known as Ebola virus disease (EVD), is a viral

Revised on 27 April 2020, haemorrhagic fever of human and other primates caused by Accepted on 18 May 2020

DOI: 10.20959/wjpr20206-17705 . Signs and symptoms typically start between two days and three weeks after contracting the virus with a fever, sore

throat, muscular pain, and headaches. Vomiting, diarrhoea and rash *Corresponding Author usually follow, along with decreased function of the liver and kidneys. Kushal Nandi

Department of At this time, some people begin to bleed both internally and

Pharmaceutical Chemistry, externally. The disease has a high risk of death, killing 25% to 90% of School of Pharmacy, those infected, with an average of about 50%. This is often due to low Techno India University, blood pressure from fluid loss, and typically follows six to 16 days Salt Lake City, Sector-V, after symptoms appear. The disease was first identified in 1976, in two EM-4, Kolkata-700091, West Bengal, India. simultaneous outbreaks: one in Nzara (a town in South ) and the other in (Democratic Republic of the Congo), a village near the from which the disease takes its name. EVD outbreaks occur intermittently in tropical regions of sub-Saharan Africa. Between 1976 and 2013, the World Health Organization reports 24 outbreaks involving 2,387 cases with 1,590 deaths. The largest outbreak to date was the in West Africa, which occurred from December 2013 to January 2016, with 28,646 cases and 11,323 deaths. It was declared no longer an emergency on 29 March 2016. Other outbreaks in Africa began in the Democratic Republic of the Congo in May 2017, and 2018. In July 2019, the World Health Organization declared the Congo Ebola outbreak a world health emergency.

KEYWORDS: Virion, Fruit bat, Nucleocapsid, Monoclonal antibodies.

www.wjpr.net Vol 9, Issue 6, 2020. 1265 Nandi et al. World Journal of Pharmaceutical Research

INTRODUCTION The virus spreads through direct contact with body fluids, such as blood from infected humans or other animals. Spread may also occur from contact with items recently contaminated with bodily fluids. Spread of the disease through the air between primates, including humans, has not been documented in either laboratory or natural conditions. Semen or breast milk of a person after recovery from EVD may carry the virus for several weeks to months. Fruit bats are believed to be the normal carrier in nature, able to spread the virus without being affected by it. Other diseases such as malaria, cholera, typhoid fever, meningitis and other viral haemorrhagic fevers may resemble EVD. Blood samples are tested for viral RNA, viral antibodies or for the virus itself to confirm the diagnosis.

Figure 1: Nucleocapsid of Ebola virion.

Control of outbreaks requires coordinated medical services and community engagement. This includes rapid detection, of those who have been exposed, quick access to laboratory services, care for those infected, and proper disposals of the dead through cremation or burial. Samples of body fluids and tissues from people with the disease should be handled with special caution. Prevention includes limiting the spread of disease from infected animals to humans by handling potentially infected only while wearing protective clothing, and by thoroughly cooking bushmeat before eating it. It also includes wearing proper protective clothing and washing hands when around a person with the disease. An was approved in the United States in December 2019. While there is no approved treatment for Ebola as of 2019, two treatments (REGN- EB3 and mAb114) are associated with improved outcomes. Supportive efforts also improve www.wjpr.net Vol 9, Issue 6, 2020. 1266 Nandi et al. World Journal of Pharmaceutical Research

outcomes. This includes either oral rehydration (drinking slightly sweetened and salty water) or giving intravenous fluids as well as treating symptoms.

Molecular genomics: Phylogenetic tree comparing ebolaviruses and . Numbers indicate percent confidence of branches. EBOV carries a negative-sense RNA genome in virions that are cylindrical/tubular, and contain viral envelope, matrix, and nucleocapsid components. The overall cylinders are generally approximately 80 nm in diameter, and have a virally encoded glycoprotein (GP) projecting as 7–10 nm long spikes from its lipid bilayer surface. The cylinders are of variable length, typically 800 nm, but sometimes up to 1000 nm long. The outer viral envelope of the virion is derived by budding from domains of host cell membrane into which the GP spikes have been inserted during their biosynthesis. Individual GP molecules appear with spacings of about 10 nm. Viral proteins VP40 and VP24 are located between the envelope and the nucleocapsid, in the matrix space. At the center of the virion structure is the nucleocapsid, which is composed of a series of viral proteins attached to an 18–19 kb linear, negative-sense RNA without 3′- polyadenylation or 5′-capping; the RNA is helically wound and complexed with the NP, VP35, VP30, and L proteins; this helix has a diameter of 80 nm.

Figure-2: Phylogenetic tree. www.wjpr.net Vol 9, Issue 6, 2020. 1267 Nandi et al. World Journal of Pharmaceutical Research

The overall shape of the virions after purification and visualization (e.g., by ultracentrifugation and electron microscopy, respectively) varies considerably; simple cylinders are far less prevalent than structures showing reversed direction, branches, and loops (e.g., U-, shepherd's crook-, 9-, or eye bolt-shapes, or other or circular/coiled appearances), the origin of which may be in the laboratory techniques applied. The characteristic "threadlike" structure is, however, a more general morphologic characteristic of filoviruses (alongside their GP-decorated viral envelope, RNA nucleocapsid, etc.).

Figure 3: Ebolaviruses and with host [fruit bat].

Genus inclusion criteria: A virus of the family is a member of the genus if its genome has several gene overlaps its fourth gene (GP) encodes four

proteins (sGP, ssGP, Δ-peptide, and GP1,2) using cotranscriptional editing to express ssGP

and GP1,2 and proteolytic cleavage to express sGP and Δ-peptide peak infectivity of its virions is associated with particles ≈805 nm in length its genome differs from that of virus by ≥50% and from that of ebolavirus by <50% at the nucleotide level its virions show almost no antigenic cross reactivity with Marburg virions.[1]

www.wjpr.net Vol 9, Issue 6, 2020. 1268 Nandi et al. World Journal of Pharmaceutical Research

Types: The genera Ebolavirus and Marburgvirus were originally classified as the species of the now-obsolete genus Filovirus. In March 1998, the Vertebrate Virus Subcommittee proposed in the International Committee on Taxonomy of Viruses (ICTV) to change the genus Filovirus to the family Filoviridae with two specific genera: Ebola-like viruses and Marburg-like viruses. This proposal was implemented in Washington, D.C., as of April 2001 and in Paris as of July 2002. In 2000, another proposal was made in Washington, D.C., to change the "-like viruses" to "-virus" resulting in today's Ebolavirus and Marburgvirus.

The five characterised species of the genus Ebolavirus are 1. (ZEBOV): Also known simply as the Zaire virus, ZEBOV has the highest case-fatality rate, up to 90% in some , with an average of approximately 83% over 27 years. There have been more outbreaks of Zaire ebolavirus than of any other species. The first outbreak took place on 26 August 1976 in Yambuku. Mabalo Lokela, a 44-year-old schoolteacher, became the first recorded case. The symptoms resembled malaria, and subsequent patients received quinine. Transmission has been attributed to reuse of unsterilized needles and close personal contact. The virus is responsible for the 2014 West Africa Ebola virus outbreak, with the largest number of deaths to date. 2. (SUDV): Like ZEBOV, SUDV emerged in 1976; it was at first assumed to be identical with ZEBOV. SUDV is believed to have broken out first amongst cotton factory workers in Nzara, Sudan (now in ), in June 1976, with the first case reported as a worker exposed to a potential . Scientists tested local animals and insects in response to this; however, none tested positive for the virus. The carrier is still unknown. The lack of barrier nursing (or "bedside isolation") facilitated the spread of the disease. The average fatality rates for SUDV were 54% in 1976, 68% in 1979, and 53% in 2000 and 2001. 3. Reston ebolavirus (RESTV): This virus was discovered during an outbreak of simian hemorrhagic fever virus (SHFV) in crab-eating macaques from Hazleton Laboratories (now Covance) in 1989. Since the initial outbreak in Reston, Virginia, it has since been found in nonhuman primates in Pennsylvania, Texas, and Siena, Italy. In each case, the affected animals had been imported from a facility in the Philippines, where the virus has also infected pigs. Despite its status as a Level-4 organism and its apparent pathogenicity in monkeys, RESTV did not cause disease in exposed human laboratory workers.

www.wjpr.net Vol 9, Issue 6, 2020. 1269 Nandi et al. World Journal of Pharmaceutical Research

4. Taï Forest ebolavirus (TAFV): Formerly known as "Côte d'Ivoire ebolavirus", it was first discovered among chimpanzees from the Tai Forest in Côte d'Ivoire, Africa, in 1994. Necropsies showed blood within the heart to be brown; no obvious marks were seen on the organs; and one necropsy displayed lungs filled with blood. Studies of tissues taken from the chimpanzees showed results similar to human cases during the 1976 Ebola outbreaks in Zaire and Sudan. As more dead chimpanzees were discovered, many tested positive for Ebola using molecular techniques. The source of the virus was believed to be the meat of infected western red colobus monkeys (Procolobus badius) upon which the chimpanzees preyed. One of the scientists performing the necropsies on the infected chimpanzees contracted Ebola. She developed symptoms similar to those of approximately a week after the necropsy, and was transported to Switzerland for treatment. She was discharged from hospital after two weeks and had fully recovered six weeks after the infection. 5. (BDBV): On November 24, 2007, the Ministry of Health confirmed an outbreak of Ebola in the Bundibugyo District. After confirmation of samples tested by the United States National Reference Laboratories and the CDC, the World Health Organization confirmed the presence of the new species. On 20 February 2008, the Uganda Ministry officially announced the end of the epidemic in Bundibugyo, with the last infected person discharged on 8 January 2008. An epidemiological study conducted by WHO and Uganda Ministry of Health scientists determined there were 116 confirmed and probable cases of the new Ebola species, and that the outbreak had a mortality rate of 34% (39 deaths).[2]

SIGNS AND SYMPTOMS Onset: The length of time between exposure to the virus and the development of symptoms (incubation period) is between two and 21 days, and usually between four and ten days. However, recent estimates based on mathematical models predict that around 5% of cases may take greater than 21 days to develop. Symptoms usually begin with a sudden influenza-like stage characterised by feeling tired, fever, weakness, decreased appetite, muscular pain, joint pain, headache, and sore throat. The fever is usually higher than 38.3°C (101°F). This is often followed by nausea, vomiting, diarrhoea, abdominal pain, and sometimes hiccups. The combination of severe vomiting and diarrhoea often leads to severe dehydration. Next, shortness of breath and may occur, along with swelling, headaches, and confusion. In about half of the cases, the skin may develop

www.wjpr.net Vol 9, Issue 6, 2020. 1270 Nandi et al. World Journal of Pharmaceutical Research

a maculopapular rash, a flat red area covered with small bumps, five to seven days after symptoms begin.

Figure 4: Signs and symptoms of Ebola.

Bleeding: In some cases, internal and external bleeding may occur. This typically begins five to seven days after the first symptoms. All infected people show some decreased blood clotting. Bleeding from mucous membranes or from sites of needle punctures has been reported in 40–50% of cases. This may cause vomiting blood, coughing up of blood, or blood in stool. Bleeding into the skin may create petechiae, purpura, ecchymoses or haematomas (especially around needle injection sites). Bleeding into the whites of the eyes may also occur. Heavy bleeding is uncommon; if it occurs, it is usually in the gastrointestinal tract. The incidence of bleeding into the gastrointestinal tract was reported to be ~58% in the 2001 outbreak in Gabon, but in the 2014-15 outbreak in the US it was ~18%, possibly due to improved prevention of disseminated.

Cause: EVD in humans is caused by four of five viruses of the genus Ebolavirus. The four are Bundibugyo virus (BDBV), Sudan virus (SUDV), Taï Forest virus (TAFV) and one simply called Ebola virus (EBOV, formerly Zaire Ebola virus). EBOV, species Zaire ebolavirus, is the most dangerous of the known EVD-causing viruses, and is responsible for the largest number of outbreaks. The fifth virus, (RESTV), is not thought to

www.wjpr.net Vol 9, Issue 6, 2020. 1271 Nandi et al. World Journal of Pharmaceutical Research

cause disease in humans, but has caused disease in other primates. All five viruses are closely related to marburgviruses.[3]

Virology

Figure 5: Electron micrograph of an Ebola virus virion.

Ebolaviruses contain single-stranded, non-infectious RNA genomes. Ebolavirus genomes contain seven genes including 3'-UTR-NP-VP35-VP40-GP-VP30-VP24-L-5'-UTR. The genomes of the five different ebolaviruses (BDBV, EBOV, RESTV, SUDV and TAFV) differ in sequence and the number and location of gene overlaps. As with all filoviruses, ebolavirus virions are filamentous particles that may appear in the shape of a shepherd's crook, of a "U" or of a "6," and they may be coiled, toroid or branched. In general, ebolavirions are 80 nanometers (nm) in width and may be as long as 14,000 nm.

Their life cycle is thought to begin with a virion attaching to specific cell-surface receptors such as C-type lectins, DC-SIGN, or integrins, which is followed by fusion of the viral envelope with cellular membranes. The virions taken up by the cell then travel to acidic endosomes and lysosomes where the viral envelope glycoprotein GP is cleaved. This processing appears to allow the virus to bind to cellular proteins enabling it to fuse with internal cellular membranes and release the viral nucleocapsid. The Ebolavirus structural glycoprotein (known as GP1,2) is responsible for the virus' ability to bind to and infect targeted cells. The viral RNA polymerase, encoded by the L gene, partially uncoats the nucleocapsid and transcribes the genes into positive-strand mRNAs, which are then translated into structural and non-structural proteins. The most abundant protein produced is the www.wjpr.net Vol 9, Issue 6, 2020. 1272 Nandi et al. World Journal of Pharmaceutical Research

nucleoprotein, whose concentration in the host cell determines when L switches from gene transcription to genome replication. Replication of the viral genome results in full-length, positive-strand antigenomes that are, in turn, transcribed into genome copies of negative- strand virus progeny. Newly synthesised structural proteins and genomes self-assemble and accumulate near the inside of the cell membrane. Virions bud off from the cell, gaining their envelopes from the cellular membrane from which they bud. The mature progeny particles then infect other cells to repeat the cycle. The genetics of the Ebola virus are difficult to study because of EBOV's virulent characteristics.[4]

Transmission

Figure 6: Life cycles of the Ebolavirus. www.wjpr.net Vol 9, Issue 6, 2020. 1273 Nandi et al. World Journal of Pharmaceutical Research

Figure 7: An illustration of safe burial practices.

It is believed that between people, Ebola disease spreads only by direct contact with the blood or other body fluids of a person who has developed symptoms of the disease. Body fluids that may contain Ebola viruses include saliva, mucus, vomit, feces, sweat, tears, breast milk, urine and semen. The WHO states that only people who are very sick are able to spread Ebola disease in saliva, and whole virus has not been reported to be transmitted through sweat. Most people spread the virus through blood, feces and vomit. Entry points for the virus include the nose, mouth, eyes, open wounds, cuts and abrasions. Ebola may be spread through large droplets; however, this is believed to occur only when a person is very sick. This contamination can happen if a person is splashed with droplets. Contact with surfaces or objects contaminated by the virus, particularly needles and syringes, may also transmit the infection. The virus is able to survive on objects for a few hours in a dried state, and can survive for a few days within body fluids outside of a person. The Ebola virus may be able to persist for more than three months in the semen after recovery, which could lead to infections via sexual intercourse. Virus persistence in semen for over a year has been recorded in a national screening programme. Ebola may also occur in the breast milk of women after recovery, and it is not known when it is safe to breastfeed again. The virus was also found in the eye of one patient in 2014, two months after it was cleared from his blood. Otherwise, people who have recovered are not infectious.

The potential for widespread infections in countries with medical systems capable of observing correct medical isolation procedures is considered low. Usually when someone has symptoms of the disease, they are unable to travel without assistance. Dead bodies remain infectious; thus, people handling human remains in practices such as traditional burial rituals or more modern processes such as embalming are at risk. 69% of the cases of Ebola

www.wjpr.net Vol 9, Issue 6, 2020. 1274 Nandi et al. World Journal of Pharmaceutical Research

infections in Guinea during the 2014 outbreak are believed to have been contracted via unprotected (or unsuitably protected) contact with infected corpses during certain Guinean burial rituals.[5]

Health-care workers treating people with Ebola are at greatest risk of infection. The risk increases when they do not have appropriate protective clothing such as masks, gowns, gloves and eye protection; do not wear it properly; or handle contaminated clothing incorrectly. This risk is particularly common in parts of Africa where the disease mostly occurs and health systems function poorly. There has been transmission in hospitals in some African countries that reuse hypodermic needles. Some health-care centres caring for people with the disease do not have running water. In the United States the spread to two medical workers treating infected patients prompted criticism of inadequate training and procedures.

Human-to-human transmission of EBOV through the air has not been reported to occur during EVD outbreaks, and airborne transmission has only been demonstrated in very strict laboratory conditions, and then only from pigs to primates, but not from primates to primates. Spread of EBOV by water, or food other than bushmeat, has not been observed. No spread by mosquitos or other insects has been reported. Other possible methods of transmission are being studied. Airborne transmission among humans is theoretically possible due to the presence of Ebola virus particles in saliva, which can be discharged into the air with a cough or sneeze, but observational data from previous epidemics suggests the actual risk of airborne transmission is low. A number of studies examining airborne transmission broadly concluded that transmission from pigs to primates could happen without direct contact because, unlike humans and primates, pigs with EVD get very high ebolavirus concentrations in their lungs, and not their bloodstream. Therefore, pigs with EVD can spread the disease through droplets in the air or on the ground when they sneeze or cough. By contrast, humans and other primates accumulate the virus throughout their body and specifically in their blood, but not very much in their lungs. It is believed that this is the reason researchers have observed pig to primate transmission without physical contact, but no evidence has been found of primates being infected without actual contact, even in experiments where infected and uninfected primates shared the same air.

Recovery and Death: Recovery may begin between seven and 14 days after first symptoms. Death, if it occurs, follows typically six to sixteen days from first symptoms and is often due to low blood pressure from fluid loss. In general, bleeding often indicates a worse www.wjpr.net Vol 9, Issue 6, 2020. 1275 Nandi et al. World Journal of Pharmaceutical Research

outcome, and blood loss may result in death. People are often in a coma near the end of life. Those who survive often have ongoing muscular and joint pain, liver inflammation, and decreased hearing, and may have continued tiredness, continued weakness, decreased appetite, and difficulty returning to pre-illness weight. Problems with vision may develop. Survivors develop antibodies against Ebola that last at least 10 years, but it is unclear whether they are immune to additional infections.

Genome: Each virion contains one molecule of linear, single-stranded, negative-sense RNA, 18,959 to 18,961 in length. The 3′ terminus is not polyadenylated and the 5′ end is not capped. This viral genome codes for seven structural proteins and one non-structural protein. The gene order is 3′ – leader – NP – VP35 – VP40 – GP/sGP – VP30 – VP24 – L – trailer – 5′; with the leader and trailer being non-transcribed regions, which carry important signals to control transcription, replication, and packaging of the viral genomes into new virions. Sections of the NP, VP35 and the L genes from filoviruses have been identified as endogenous in the genomes of several groups of small mammals.[6]

Figure-8: Genome.

It was found that 472 nucleotides from the 3' end and 731 nucleotides from the 5' end are sufficient for replication of a viral "minigenome", though not sufficient for infection. Virus sequencing from 78 patients with confirmed Ebola virus disease, representing more than 70% of cases diagnosed in Sierra Leone from late May to mid-June 2014, provided evidence that the 2014 outbreak was no longer being fed by new contacts with its natural reservoir. Using third-generation sequencing technology, investigators were able to sequence samples as quickly as 48 hours. Like other RNA viruses, Ebola virus mutates rapidly, both within a person during the progression of disease and in the reservoir among the local human population. The observed mutation rate of 2.0 × 10−3 substitutions per site per year is as fast as that of seasonal influenza.

www.wjpr.net Vol 9, Issue 6, 2020. 1276 Nandi et al. World Journal of Pharmaceutical Research

Entry

Figure-9: NPC1.

NPC1: There are two candidates for host cell entry proteins. The first is a cholesterol transporter protein, the host-encoded Niemann–Pick C1 (NPC1), which appears to be essential for entry of Ebola virions into the host cell and for its ultimate replication. In one study, mice with one copy of the NPC1 gene removed showed an 80 percent survival rate fifteen days after exposure to mouse-adapted Ebola virus, while only 10 percent of unmodified mice survived this long. In another study, small molecules were shown to inhibit Ebola virus infection by preventing viral envelope glycoprotein (GP) from binding to NPC1. Hence, NPC1 was shown to be critical to entry of this filovirus, because it mediates infection by binding directly to viral GP. When cells from Niemann–Pick Type C individuals lacking this transporter were exposed to Ebola virus in the laboratory, the cells survived and appeared impervious to the virus, further indicating that Ebola relies on NPC1 to enter cells; mutations in the NPC1 gene in humans were conjectured as a possible mode to make some individuals resistant to this deadly . The same studies described similar results regarding NPC1's role in virus entry for , a related filovirus. A further study has also presented evidence that NPC1 is the critical receptor mediating Ebola infection via its direct binding to the viral GP, and that it is the second "lysosomal" domain of NPC1 that mediates this binding.

The second candidate is TIM-1 (a.k.a. HAVCR1). TIM-1 was shown to bind to the receptor binding domain of the EBOV glycoprotein, to increase the receptivity of Vero cells. Silencing its effect with siRNA prevented infection of Vero cells. TIM1 is expressed in tissues known to be seriously impacted by EBOV lysis (trachea, cornea, and conjunctiva). A against the IgV domain of TIM-1, ARD5, blocked EBOV binding and infection. Together, these studies suggest NPC1 and TIM-1 may be potential therapeutic targets for an Ebola anti-viral drug and as a basis for a rapid field diagnostic assay.[7]

www.wjpr.net Vol 9, Issue 6, 2020. 1277 Nandi et al. World Journal of Pharmaceutical Research

Replication: Being acellular, viruses such as Ebola do not replicate through any type of cell division; rather, they use a combination of host- and virally encoded enzymes, alongside host cell structures, to produce multiple copies of themselves. These then self-assemble into viral macromolecular structures in the host cell. The virus completes a set of steps when infecting each individual cell. The virus begins its attack by attaching to host receptors through the glycoprotein (GP) surface peplomer and is endocytosed into macropinosomes in the host cell. To penetrate the cell, the viral membrane fuses with vesicle membrane, and the nucleocapsid is released into the cytoplasm. Encapsidated, negative-sense genomic ssRNA is used as a template for the synthesis (3'-5') of polyadenylated, monocistronic mRNAs and, using the host cell's ribosomes, tRNA molecules, etc., the mRNA is translated into individual viral proteins. These viral proteins are processed: a glycoprotein precursor (GP0) is cleaved to GP1 and GP2, which are then heavily glycosylated using cellular enzymes and substrates. These two molecules assemble, first into heterodimers, and then into trimers to give the surface peplomers. Secreted glycoprotein (sGP) precursor is cleaved to sGP and delta peptide, both of which are released from the cell. As viral protein levels rise, a switch occurs from translation to replication. Using the negative-sense genomic RNA as a template, a complementary +ssRNA is synthesized; this is then used as a template for the synthesis of new genomic (-)ssRNA, which is rapidly encapsidated. The newly formed nucleocapsids and envelope proteins associate at the host cell's plasma membrane; budding occurs, destroying the cell.

Ebola Vaccines: Ebola vaccines are a number of vaccines to prevent Ebola that are either approved or in development. The first vaccine to be approved in the United States was rVSV- ZEBOV in December 2019. It had been used extensively in Ebola epidemic under a compassionate use protocol. Several promising vaccine candidates have been shown to protect nonhuman primates (usually macaques) against lethal infection.

Vaccines include replication-deficient adenovirus vectors, replication-competent vesicular stomatitis (VSV) and human parainfluenza (HPIV-3) vectors, and virus-like nanoparticle preparations. Conventional trials to study efficacy by exposure of humans to the pathogen after immunization are not ethical in this case. For such situations, the Food and Drug Administration (FDA) has established the "animal efficacy rule" allowing licensure to be approved on the basis of animal model studies that replicate human disease, combined with evidence of safety and a potentially potent immune response (antibodies in the blood) from

www.wjpr.net Vol 9, Issue 6, 2020. 1278 Nandi et al. World Journal of Pharmaceutical Research

humans given the vaccine. Clinical trials involve the administration of the vaccine to healthy human subjects to evaluate the immune response, identify any side effects and determine the appropriate dosage.[8]

APPROVED VACCINES rVSV-ZEBOV: VSV-EBOV or rVSV-ZEBOV, sold under the brand name Ervebo, is a vaccine based on the vesicular stomatitis virus which was genetically modified to express a surface glycoprotein of Zaire Ebola virus. In November 2019, the European Commission granted a conditional marketing authorization. The WHO prequalification came fewer than 48 hours later, making it the fastest vaccine prequalification process ever conducted by WHO. In December 2019, it was approved for medical use in the United States.

It was developed by the Agency of , with development subsequently taken over by Merck Inc. In October 2014, the Wellcome Trust, who was also one of the biggest UK founders, announced the start of multiple trials in healthy volunteers in Europe, Gabon, Kenya, and the US. The vaccine was proven safe at multiple sites in North America, Europe, and Africa, but several volunteers at one trial site in Geneva, Switzerland, developed vaccine-related arthritis after about two weeks, and about 20–30% of volunteers at reporting sites developed low-grade post-vaccine fever, which resolved within a day or two. Other common side-effects were pain at the site of injection, , and fatigue. The trial was temporarily halted in December 2014 due to possible adverse effects, but subsequently resumed. As of April 2015, a Phase III trial with a single dose of VSV-EBOV began in Liberia after a successful Phase II study in the West African country. On 31 July 2015, preliminary results of a Phase III trial in Guinea indicated that the vaccine appears to be "highly efficacious and safe." The trial used a protocol that first vaccinated all the closest contacts of new cases of Ebola infection either immediately or after 21 days. Because of the demonstrated efficacy of immediate vaccination, all recipients will now be immunized immediately. Ring vaccination is the method used in the program to eradicate smallpox in the 1970s. The trial will continue to assess whether the vaccine is effective in creating herd immunity to Ebola virus infection. In December 2016, a study found the VSV-EBOV vaccine to be 95–100% effective against the Ebola virus, making it the first proven vaccine against the disease.

The approval was supported by a study conducted in Guinea during the 2014–2016 outbreak in individuals 18 years of age and older. The study was a randomized cluster (ring) www.wjpr.net Vol 9, Issue 6, 2020. 1279 Nandi et al. World Journal of Pharmaceutical Research

vaccination study in which 3,537 contacts, and contacts of contacts, of individuals with laboratory-confirmed Ebola virus disease (EVD) received either "immediate" or 21-day "delayed" vaccination. This design was intended to capture a social network of individuals and locations that might include dwellings or workplaces where a patient spent time while symptomatic, or the households of individuals who had contact with the patient during that person's illness or death. In a comparison of cases of EVD among 2,108 individuals in the "immediate" vaccination arm and 1,429 individuals in the "delayed" vaccination arm, Ervebo was determined to be 100% effective in preventing Ebola cases with symptom onset greater than ten days after vaccination. No cases of EVD with symptom onset greater than ten days after vaccination were observed in the "immediate" cluster group, compared with ten cases of EVD in the 21-day "delayed" cluster group.

In additional studies, antibody responses were assessed in 477 individuals in Liberia, approximately 500 individuals in Sierra Leone and approximately 900 individuals in Canada, Spain and the US. The antibody responses among those in the study conducted in Canada, Spain and the US were similar to those among individuals in the studies conducted in Liberia and Sierra Leone. The safety was assessed in approximately 15,000 individuals in Africa, Europe and North America. The most commonly reported side effects were pain, swelling and redness at the injection site, as well as headache, fever, joint and muscle aches and fatigue. In December 2016, a study found the VSV-EBOV vaccine to be 70–100% effective against the Ebola virus, making it the first proven vaccine against the disease. However, the design of this study and the high efficacy of the vaccine were questioned. In November 2019, the European Commission granted a conditional marketing authorization to Ervebo (rVSV∆G-ZEBOV-GP, live) and the WHO prequalified an Ebola vaccine for the first time.[9]

VACCINES IN PIPELINE Table 1: List of vaccines. Associated Vaccine Status organisations Chimp adenovirus 3 vectored GSK & NIAID Phase III Feb. 2016 glycoprotein (cAd3-EBO Z) rVSV vectored glycoprotein (VSV- Newlink Genetics In use EBOV) & Merck Human adenovirus 5 vectored 2014 BIT & CanSino Phase I complete glycoprotein insert (Ad5-EBOV) Adenovirus 26 vectored glycoprotein / Phase I complete Johnson & Johnson MVA-BN (Ad26.ZEBOV/MVA-BN) April, 2016

www.wjpr.net Vol 9, Issue 6, 2020. 1280 Nandi et al. World Journal of Pharmaceutical Research

Ministry of Health HPIV-3 vectored glycoprotein Phase I planned (Russia) Thomas Jefferson Non-human primate vectored glycoprotein University & NIAID challenge complete NHP challenge Purified glycoprotein Protein Sciences initiated Non-human primate Ebola ∆VP30 H O treated University of Wisconsin 2 2 challenge complete

cAd3-EBO Z

Figure-10: Monoclonal antibodies.

Ebola virions: In September 2014, two Phase I clinical trials began for the vaccine cAd3- EBO Z, which is based on an attenuated version of a chimpanzee adenovirus (cAd3) that has been genetically altered so that it is unable to replicate in humans. The cAd3 vector has a DNA fragment insert that encodes the Ebola virus glycoprotein, which is expressed on the virion surface and is critical for attachment to host cells and catalysis of membrane fusion. It was developed by NIAID in collaboration with Okairos, now a division of GlaxoSmithKline. For the trial designated VRC 20, 20 volunteers were recruited by the NIAID in Bethesda, Maryland, while three dose-specific groups of 20 volunteers each were recruited for trial EBL01 by University of Oxford, UK. Initial results were released in November 2014; all 20 volunteers developed antibodies against Ebola and there were no significant concerns raised about safety. In December 2014, University of Oxford expanded the trial to include a booster vaccine based on MVA-BN, a strain of Modified vaccinia Ankara, developed by Bavarian Nordic, to investigate whether it can help increase immune responses further. The trial which has enrolled a total of 60 volunteers will see 30 volunteers vaccinated with the booster vaccine. As of April 2015, Phase III trial with a single dose of cAd3-EBO Z begins in Sierra Leone after a successful Phase 2 study in West Africa countries.[10]

Ad26.ZEBOV/MVA-BN: Johnson & Johnson developed an Ebola vaccine at its Janssen Pharmaceutical Company. The regimen consists of two vaccine components (first vaccine as www.wjpr.net Vol 9, Issue 6, 2020. 1281 Nandi et al. World Journal of Pharmaceutical Research

prime, followed by a second vaccine as boost) that are based on AdVac technology from Crucell Holland B.V., which is part of Janssen, and the MVA-BN technology from Bavarian Nordic. The Ad26.ZEBOV is derived from human adenovirus serotype 26 (Ad26) expressing the Ebola virus Mayinga variant glycoprotein while the second component MVA-BN is the Modified Vaccinia Virus Ankara – Bavarian Nordic (MVA-BN) Filo- vector. This product commenced Phase I at the Jenner Institute in Oxford during January 2015. The preliminary data indicated the prime-boost vaccine regimen elicited temporary immunologic response in the volunteers as expected from vaccination. The Phase II trial enrolled 612 adult volunteers and commenced in July 2015, in the and . A second Phase II trial, involving 1,200 volunteers, was initiated in Africa with the first trial commenced in Sierra Leone in October 2015.

In September 2019, the Committee for Medicinal Products for Human Use (CHMP) of the European Agency (EMA) granted an accelerated assessment to Janssen for Ad26.ZEBOV and MVA-BN, and in November 2019, Janssen submitted a Marketing Authorization Application (MAA) to the EMA for approval of Ad26.ZEBOV and MVA-BN.

Ebola GP vaccine: Recombinant formation plasmids: At the 8th Vaccine and ISV Conference in Philadelphia on 27−28 October 2014, Novavax Inc. reported the development in a "few weeks" of a glycoprotein (GP) nanoparticle Ebola virus (EBOV GP) vaccine using their proprietary recombinant technology. A recombinant protein is a protein whose code is carried by recombinant DNA. The vaccine is based on the newly published genetic sequence of the 2014 Guinea Ebola (Makona) strain that is responsible for the 2014 Ebola disease epidemic in West Africa. In animal studies, a useful immune response was induced, and was found to be enhanced ten to a hundred-fold by the company's "Matrix- M" immunologic adjuvant. A study of the response of non-human primate to the vaccine had been initiated. As of February 2015, Novavax had completed two primate studies on baboons and macaques and had initiated a Phase I clinical trial in Australia. The Lipid nanoparticle (LNP)-encapsulated siRNAs rapidly adapted to target the Makona outbreak strain of EBOV and are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at three days post-exposure while animals were viremic and clinically ill. The top line Phase I human trial results showed that the adjuvanted Ebola GP Vaccine was highly immunogenic at all dose levels.[11]

www.wjpr.net Vol 9, Issue 6, 2020. 1282 Nandi et al. World Journal of Pharmaceutical Research

Nasal vaccine: On 5 November 2014, the Houston Chronicle reported that a research team at the University of Texas-Austin was developing a nasal spray Ebola vaccine, which the team had been working on for seven years. The team reported in 2014, that in the nonhuman primate studies it conducted, the vaccine had more efficacy when delivered via nasal spray than by injection. As of November 2014, further development by the team appeared unlikely due to lack of funding.

Figure-11: Nasal spray vaccine.

Ad5-EBOV: In late 2014 and early 2015, a double-blind, randomized Phase I trial was conducted in the Jiangsu Province of China; the trial examined a vaccine that contains glycoproteins of the 2014 strain, rather than those of the 1976 strain. The trial found signals of efficacy and raised no significant safety concerns.

In 2017, the China Food and Drug Administration (CFDA) announced approval of an Ebola vaccine, co-developed by the Institute of Biotechnology of the Academy of Military Medical Sciences and the private vaccine-maker CanSino Biologics. It contains a human adenovirus serotype 5 vector (Ad5) with the glycoprotein gene from ZEBOV. Their findings were consistent with previous tests on rVSV-ZEBOV in Africa and Europe.

Vaxart tablet: Vaxart Inc. is developing a vaccine technology in the form of a temperature- stable tablet which may offer advantages such as reduced cold chain requirement, and rapid and scalable manufacturing. In January 2015, Vaxart announced that it had secured funding to develop its Ebola vaccine to Phase I trial.

Attenuated Ebola Virus vaccine: A study published in Science during March 2015, demonstrated that vaccination with a weakened form of the Ebola virus provides some measure of protection to non-human primates. This study was conducted in accordance with a protocol approved by an Institutional Animal Care and Use Committee of the National

www.wjpr.net Vol 9, Issue 6, 2020. 1283 Nandi et al. World Journal of Pharmaceutical Research

Institutes of Health. The new vaccine relies on a strain of Ebola called EBOVΔVP30, which is unable to replicate.

GamEvac-Combi: A study published in Human Vaccines & Immunotherapeutics in March 2017, analyzing data from a clinical trial of the GamEvac-Combi vaccine in Russia, concluded said vaccine to be safe and effective and recommended proceeding to Phase III trials.

Prospects: In September 2019, a study published in Cell Reports demonstrated the role of the Ebola virus VP35 protein in its immune evasion. A recombinant form of Ebola virus with a mutant VP35 protein (VP35m) was developed, and showed positive results in the activation of the RIG-I-like receptor signaling. Non-human primates were challenged with different doses of VP35. This challenge resulted in the activation of the innate immune system and the production of anti-EBOV antibodies. The primates were then back-challenged with the wild type Ebola virus and survived. This potentially creates a prospect for a future vaccine development.

Clinical trials in West Africa: In January 2015, Marie-Paule Kieny, the World Health Organization's (WHO) assistant director-general of health systems and innovation, announced that the vaccines cAd3-EBO Z and VSV-EBOV had demonstrated acceptable safety profiles during early testing and would soon progress to large-scale trials in Liberia, Sierra Leone and Guinea. The trials would involve up to 27,000 people and comprise three groups – members of the first two groups would receive the two candidate vaccines, while the third group will receive a placebo. Both vaccines have since successfully completed the Phase 2 studies. The large-scale Phase 3 studies have begun as of April 2015, in Liberia and Sierra Leone, and in Guinea in March 2016.

In addition, a medical anthropologist at Université de Montréal, had been working in Guinea and raised further questions about safety in the ring trial after spending time in April at one of the Ebola treatment units where trial participants are taken if they become ill, the centre in Coyah, about 50 km from the capital of Conakry.

The Russian Foreign Ministry announced in 2016, the intention to conduct field trials of two Russian vaccines involving 2000 people. According to local media reports, the Guinean government authorized the commencement of the trials on 9 August 2017, at the Rusal-built

www.wjpr.net Vol 9, Issue 6, 2020. 1284 Nandi et al. World Journal of Pharmaceutical Research

Research and Diagnostic Center of Epidemiology and Microbiology in Kindia. The trials were slated to continue until 2018. As of October 2019, Russia licensed the vaccine and was reportedly ready to ship vaccine to Africa.[12]

U.S. national stockpile: In 2014, Credit Suisse estimated that the U.S. government will provide over $1 billion in contracts to companies to develop and vaccines for Ebola virus disease. Congress passed a law in 2004, that funds a national stockpile of vaccines and medicine for possible outbreaks of disease. A number of companies were expected to develop Ebola vaccines: GlaxoSmithKline, NewLink Genetics, Johnson & Johnson and Bavarian Nordic. Another company, Emergent BioSolutions, was a contestant for manufacturing new doses of ZMapp, a drug for Ebola virus disease treatment originally developed by Mapp Biopharmaceutical. Supplies of ZMapp ran out in August 2014. In September 2014, the Biomedical Advanced Research and Development Authority (BARDA) entered into a multimillion-dollar contract with Mapp Biopharmaceutical to accelerate the development of ZMapp. Additional contracts were signed in 2017.

CONCLUSION Numbers of monoclonal antibodies have been launched to combat over the deadly virions of Ebola virus which are mainly glycoproteins in nature made of carbohydrate+polypeptide. These can act on glycoprotein and nucleoprotein of Ebola genomics to stop the replication on nucleocapsid of Ebola virion by competitive inhibition of nucleic acid of RNA leading to cell death. Proteolytic cleavage of RNA polymerase of Ebola virion is ruptured to produce minigenome by replication of nucleic acid is blocked to produce multiphase virions. Human- to-human transmission of the Ebola virus is primarily associated with direct or indirect contact with blood and body fluids. Transmission to healthcare workers has been reported when appropriate infection control measures have not been observed. It is not always possible to identify patients with EBV early because initial symptoms may be nonspecific. For this reason, it is important that healthcare workers apply standard precautions consistently with all patients – regardless of their diagnosis – in all work practices at all times. These include basic hand hygiene, respiratory hygiene, the use of personal protective equipment (according to the risk of splashes or other contact with infected materials), safe injection practices and safe burial practices. Health-care workers caring for patients with suspected or confirmed Ebola virus should apply, in addition to standard precautions, other infection control measures to avoid any exposure to the patient’s blood and body fluids and direct unprotected contact with

www.wjpr.net Vol 9, Issue 6, 2020. 1285 Nandi et al. World Journal of Pharmaceutical Research

the possibly contaminated environment. When in close contact (within 1 metre) of patients with EBV, health-care workers should wear face protection by face shield or a medical mask and goggles, a clean, non-sterile long-sleeved gown and gloves (sterile gloves for some procedures). Laboratory workers are also at risk. Samples taken from suspected human and animal Ebola cases for diagnosis should be handled by trained staff and processed in suitably equipped laboratories.

REFERENCES 1. Patel, K.S., Patel, A.K.; Chaudhary, H.H; Aal, J.B.; Parmar, J.N.; Patel, V.R and Sen, D.J.; Pathogenesis of ebola virus: a deadly virion hosted by bats: World Journal of Pharmaceutical Sciences, 2014; 2(9): 1097-1113. 2. Na, Woonsung; Park, Nanuri; Yeom, Minju; Song, Daesub. "Ebola outbreak in Western Africa 2014: what is going on with Ebola virus?". Clinical and Experimental Vaccine Research, 2016; 4(1): 17–22. 3. Pattyn, S.; Jacob, W.; van der Groen, G.; Piot, P.; Courteille, G. "Isolation of Marburg- like virus from a case of haemorrhagic fever in Zaire". Lancet, 1977; 309(8011): 573–4. 4. Bowen, E. T. W.; Lloyd, G.; Harris, W. J.; Platt, G. S.; Baskerville, A.; Vella, E. E. "Viral haemorrhagic fever in southern Sudan and northern Zaire. Preliminary studies on the aetiological agent". Lancet, 1977; 309(8011): 571–3. 5. Hoenen T, Groseth A, Feldmann H. "Current ebola vaccines". Expert Opinion on Biological Therapy, 2012; 12(7): 859–72. 6. Peterson AT, Bauer JT, Mills JN. "Ecologic and geographic distribution of filovirus disease". Emerging Infectious Diseases, 2004; 10(1): 40–47. 7. Fausther-Bovendo H, Mulangu S, Sullivan NJ. "Ebolavirus vaccines for humans and apes". Current Opinion in Virology, 2012; 2(3): 324–29. 8. Pavot V. "Ebola virus vaccines: Where do we stand?". Clinical , 2016; 173: 44–49. 9. Marzi A, Hanley PW, Haddock E, Martellaro C, Kobinger G, Feldmann H. "Efficacy of Vesicular Stomatitis Virus-Ebola Virus Postexposure Treatment in Rhesus Macaques Infected With Ebola Virus Makona". The Journal of Infectious Diseases, 2016; 214(3): S360–66. 10. Marzi A, Robertson SJ, Haddock E, Feldmann F, Hanley PW, Scott DP, et al. "Ebola Vaccine. VSV-EBOV rapidly protects macaques against infection with the 2014/15 Ebola virus outbreak strain". Science, 2015; 349(6249): 739–42. www.wjpr.net Vol 9, Issue 6, 2020. 1286 Nandi et al. World Journal of Pharmaceutical Research

11. Kuhn, J. H.; Becker, S.; Ebihara, H.; Geisbert, T. W.; Johnson, K. M.; Kawaoka, Y.; Lipkin, W. I.; Negredo, A. I.; Netesov, S. V.; Nichol, S. T.; Palacios, G.; Peters, C. J.; Tenorio, A.; Volchkov, V. E.; Jahrling, P. B. "Proposal for a revised taxonomy of the family Filoviridae: Classification, names of taxa and viruses, and virus abbreviations". Archives of Virology, 2010; 155(12): 2083–103. 12. Pringle, C. R. "Virus taxonomy-San Diego". Archives of Virology, 1998; 143(7): 1449–59.

www.wjpr.net Vol 9, Issue 6, 2020. 1287