Recent advances in the development of vaccines for Ebola virus disease

Virus Research 211 (2016) 174–185

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Virus Research journal homepage: www.elsevier.com/locate/virusres

Review

Recent advances in the development of vaccines for Ebola virus disease Elijah Ige Ohimain Medical and Public Health Microbiology Research Unit, Biological Sciences Department, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria

a r t i c l e

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Article history: Received 29 June 2015 Received in revised form 11 October 2015 Accepted 16 October 2015 Available online 24 October 2015 Keywords: Hemorrhagic fever Pre-existing immunity Replication competent vaccine Replication incompetent Vaccine Zoonotic infections

a b s t r a c t Ebola virus is one of the most dangerous microorganisms in the world causing hemorrhagic fevers in humans and non-human primates. Ebola virus (EBOV) is a zoonotic infection, which emerges and reemerges in human populations. The 2014 outbreak was caused by the Zaire strain, which has a kill rate of up to 90%, though 40% was recorded in the current outbreak. The 2014 outbreak is larger than all 20 outbreaks that have occurred since 1976, when the virus was first discovered. It is the first time that the virus was sustained in urban centers and spread beyond Africa into Europe and USA. Thus far, over 22,000 cases have been reported with about 50% mortality in one year. There are currently no approved therapeutics and preventive vaccines against Ebola virus disease (EVD). Responding to the devastating effe1cts of the 2014 outbreak and the potential risk of global spread, has spurred research for the development of therapeutics and vaccines. This review is therefore aimed at presenting the progress of vaccine development. Results showed that conventional inactivated vaccines produced from EBOV by heat, formalin or gamma irradiation appear to be ineffective. However, novel vaccines production techniques have emerged leading to the production of candidate vaccines that have been demonstrated to be effective in preclinical trials using small animal and non-human primates (NHP) models. Some of the promising vaccines have undergone phase 1 clinical trials, which demonstrated their safety and immunogenicity. Many of the candidate vaccines are vector based such as Vesicular Stomatitis Virus (VSV), Rabies Virus (RABV), Adenovirus (Ad), Modified Vaccinia Ankara (MVA), Cytomegalovirus (CMV), human parainfluenza virus type 3 (HPIV3) and Venezuelan Equine Encephalitis Virus (VEEV). Other platforms include virus like particle (VLP), DNA and subunit vaccines. © 2015 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Ebola virus genes and their functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Candidates vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 3.1. Replication competent vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 3.1.1. Recombinant vesicular stomatitis virus (rVSV) based vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 3.1.2. Rabies virus based vaccine platform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 3.1.3. Recombinant cytomegalovirus (rCMV) based platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 3.1.4. Human parainfluenza virus type 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 3.2. Non-replicating (replication incompetent) vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 3.2.1. Adenovirus based vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 3.2.2. DNA and subunit vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 3.2.3. Virus like particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 3.2.4. Venezuelan Equine Encephalitis virus replicon vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 3.2.5. Vaccinia based ebola vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 3.2.6. Inactivated vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

E-mail addresses: [email protected], [email protected] http://dx.doi.org/10.1016/j.virusres.2015.10.021 0168-1702/© 2015 Elsevier B.V. All rights reserved.

E.I. Ohimain / Virus Research 211 (2016) 174–185

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3.2.7. Other vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

1. Introduction Ebola virus disease (EVD) formerly called Ebola hemorrhagic fever (EHF) is caused by Ebola virus of the family Filoviridae. Ebola virus was discovered in 1976 with simultaneous outbreaks in Democratic Republic of Congo (DRC) and Sudan (Feldmann and Geisbert, 2011). Since its discovery 40 years ago, the virus has caused over 20 sporadic outbreaks mostly confined to rural areas in East and Central Africa (Patel et al., 2007). Hence the disease did not attract much global attention. But the 2014 outbreak, which emerged in West Africa, took a new and unprecedented pattern. The disease was first noticed in a 2 years old child on 6 December 2013 in a rural village in Guinea (Onwuakor, 2014) close to the borders of Liberia and Sierra Leone. From there, it appears that the virus spread within these three countries unnoticed. But officially, the World Health Organization (WHO) declared an outbreak of EVD on 22 March 2014 in Guinea, 31 March 2014 in Liberia, 26 May 2014 in Sierra Leone, 20 July in Nigeria and 29 August in Senegal. Due to increased travel mostly through air, the disease was reported in other parts of the World including US (25 October 2014), Spain (6 October 2014) and Mali (25 October 2014). Other cases were also reported in the UK. Hence what started as a West African problem soon became a global threat. Hence, on 8 August 2014, the WHO declared the epidemic as a global public health emergency, while on 18 September 2014, the United Nations Security Council (UNSC) adopted resolutions 2177, declaring the disease a threat to international peace and security. The world responded, though a little late, by sending medical personnel, equipment, supportive drugs and finance. International NGOs particularly Doctors Without Borders (MSF) and other charity organization sent their staff to combat EVD even at a time when West Africa was practically isolated. Some of these NGO staff got infected and returned home to seek medical attention, which was partially how the disease spread to Western Countries. As there were no cure, they were given experimental therapies, while some survived, a few unfortunately died. Notably among the countries that assisted West Africa against EVD was USA, UK, France, Germany, China, Japan, and Cuba. The World Bank, Africa Development Bank and Bill and Melinda Gates Foundation supported financially. By the time most of this assistance came, the disease has caused major catastrophic disaster in West Africa. As of 14 September 2014, a total of 4507 EVD cases have been reported in Liberia, Sierra Leone, Guinea, Nigeria and Senegal with 2296 deaths (WHO Ebola Response Team, 2014). The spread of Ebola virus was successfully curtailed in Senegal and Nigeria, with the WHO official declaring them free of EVD after 42 days i.e., twice the incubation period of the virus on 17 October 2014 and 20 October 2014 respectively. Current statistics from WHO show that as of 19 August 2015 a total of 27,988 persons have been infected worldwide with the following breakdown in the three main countries; Guinea (3766 infected, 2524 dead), Liberia (10,672 infected, 4808 dead) and Sierra Leone (13,494 infected, 3952 dead) (WHO, 2015). The published data might have been under reported (Choi et al., 2015; The Economist, 2015). Through international/global concerted efforts, the rate of infection is now declining. Apart from deaths, and the burden of disease, EVD has caused social challenges (Tayo et al., 2015; Chigbu and Ntiador, 2014) and economic problems (Adegun, 2014; Cheto, 2014). The infection rate may not abate in the next decade. The World Bank (2014) estimated the shortterm fiscal impacts of EVD based on sector component methods.

They found out that the impact was large, being $93 million (4.7% of GDP) for Liberia, $79 million in (1.8% of GDP) of Sierra Leone and $120 million (1.2% of GDP) for Guinea. The genus Ebolavirus consist of 5 distinct species in decreasing order of virulence; Zaire Ebola virus (ZAIV), Sudan ebolavirus (SUDV), Bundibugyo ebolavirus (BDBV or BEBOV), Tai Forest ebolavirus (TAFV) and Reston ebolavirus (RESTV) (Bukreyev et al., 2014), which do not infect human but mostly non-human primates (NHPs). Fruit bats are regarded as the primary host of the virus, from where it spreads to human directly or indirectly through intermediate reservoirs such as NHPs particularly monkeys, gorilla and baboons and other wildlife including duikers, pigs and arthropods. Among humans, Ebola virus can spread via direct contact through exchange of body fluids and secretions such as sweat, semen, blood, urine, catarrh, saliva, sputum, and vomitus. Ebola virus is spread by direct contact with infected persons or corpse during funerals. Ebola virus is also commonly spread via nosocomial infections (Shuaib et al., 2014). Though, the incubation period of the virus is 2–21 days, death usually occurs within 4–10 days. Ebola virus disease is characterized by sudden onset of fever, weakness, headache, muscle pain, sore throat, hiccups, conjunctivitis and red eyes, rash, diarrhea and vomiting, internal and external bleeding. Like HIV/AIDs, Ebola virus evade and damage the host immune system (Qiu et al., 2012, 2013; Watanabe et al., 2007; Smith et al., 2013) leading to coagulopathy resulting in multi-organ destruction including the liver and kidneys (Beeching et al., 2014; Bente et al., 2009; Martin et al., 2006). The ability of Ebola virus to interfere with the innate immunity system of the host, especially the interferon response is caused by virus matrix proteins VP24 and VP35 (Bente et al., 2009; Watanabe et al., 2007; Qiu et al., 2012, 2013). Ebola virus disease, which started as a localized problem in Africa has grown to become a global threat. Ebola virus disease is now a threat to global peace and security for several reasons. The virus is a zoonotic pathogen with outbreak occurring sporadically in Africa, emerging and re-emerging (Peters et al., 1994; Marston et al., 2014; Sarwar et al., 2015). There have been over 20 outbreaks since, the disease was first reported in 1976 (Kortepeter et al., 2011; Mire et al., 2013). According to Richardson et al. (2009) EVD has drawn increasing interest in the past few years due to increasing number of natural outbreaks especially in Africa. Zaire Ebola virus is the most aggressive/virulent species (Richardson et al., 2011; Bausch, 2014), its fatality rates have been reported to be up to 90% (Basler and Amerasinghe, 2009; Gunther et al., 2011; Marzi et al., 2011; Mire et al., 2014; Sullivan et al., 2006; Tsuda et al., 2011). Zaire Ebola virus is the cause of the 2014 EVD outbreak in West Africa (Kanapathipillai et al., 2014; Sarwar et al., 2015; Bishop, 2015) though with less fatality rate 40–87% (Ohimain, 2015). The current outbreak, which is caused by Makona outbreak strain of EBOV, is larger than all previous epidemics combined (Bishop, 2015). This is the first time that EVD is localized primarily in urban areas with a global spread (Sarwar et al., 2015). The World Health Organization classified Ebola virus as a biosafety level 4 pathogens (Bente et al., 2009; Enterlein et al., 2006; Gunther et al., 2011) though there are limited level 4 facilities in Africa, where the outbreaks occur frequently. The Center for Disease Control and prevention (CDC) classified Ebola virus as a category A pathogen that can be used as a biological weapon for bioterrorism (Richardson et al., 2009; Feldmann et al., 2007; Swenson et al.,

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2008; Sarwar et al., 2015; Patel et al., 2007; Feldmann and Geisbert, 2011; Bente et al., 2009). Moreover, the WHO estimated that about 1.4 million people could contract the disease before the current outbreak ends (Choi et al., 2015; Salaam-Blyther, 2014). Unfortunately, there are no approved therapies and vaccine for treatment and prevention of the disease. EVD is currently being managed by the use of supportive/palliative treatment, which involves treating the symptoms through oral rehydration therapy, administration of pain killers, control of blood pressure and the opportunity of infection. Certain experimental drugs were used on compassionate grounds in the current outbreak including ZMapp, TKM-Ebola, Favipiravir, BCX 4430, AVI 7537 and Brincidofovir. Serum from convalescent plasma was also used. The experimental drugs resulted in limited successes that cannot be unequivocally established. Moreover, most of them have not been previously tested in humans for safety and efficacy. Besides, the available doses are too few to be able address epidemics of this magnitude. For instance, all the 9 doses of ZMapp available have been used and more can only be produced within months. Hence, the need for pre-and post-exposure vaccines and prophylaxis that can protect the global community in the event of either bioterrorism or natural outbreak is urgent. Kanapathipillai et al. (2014) said that the development of Ebola virus vaccine is an urgent international priority. Choi et al. (2015) reported the need for long-lasting vaccines to preserve global health. Though, the devastating effect of the 2014 EVD outbreak had spurred research into drugs and vaccines, studies have shown that progress made in therapeutics is slightly slower (Feldmann and Geisbert, 2011). A vaccine that is effective, cheap and safe that can be administered in single dose is urgently being sought for. Hence, this paper’s aim is to review the progress made on the development of candidate vaccines that could possibly protect against the current Ebola virus species and emerging mutants. We start by first describing Ebola virus genes/proteins, which are the prime targets for candidate vaccines.

2. Ebola virus genes and their functions Novel therapeutic agents and potential vaccines are designed to target different steps in the replication cycle of Ebola virus (Lai et al., 2014). The genome of Ebola virus, which is about 19 kb long, consists of genes that encode for 7 proteins (Geisbert et al., 2010; Feldmann and Geisbert, 2011; Peters et al., 1994; Enterlein et al., 2006; Sobarzo et al., 2012). Four of the genes encodes for structural proteins including virion envelope glycoprotein (GP), nucleoprotein (NP) and 2 viral (matrix) proteins VP24 and VP40, while the nonstructural proteins include VP 30 and VP35, and the RNA dependent viral polymerase (L) (Sullivan et al., 2003). Several reports suggest that all 7 proteins (NP, VP35, VP40, GP, VP30, VP24, L) are structural (Lai et al., 2014; Feldmann et al., 2003; Trunschke et al., 2013; Wilson et al., 2001). The order of occurrence of the genes are as follows; 3 Leader, NP, VP35, VP40, GP, VP30, VP24, L and 5 trailer (Fieldmann and Geisbert, 2013; Geisbert et al., 2010). A simplified version of the genome is presented in Fig. 1. Most of these gene products have various functions (Table 1). For instance, the glycoprotein is responsible for binding and viral entry (Sobarzo et al., 2012; Enterlein et al., 2006; Geisbert et al., 2010; Watanabe et al., 2007), NP, VP35, VP30 and L are responsible for replication and transcription of viral RNA (Sobarzo et al., 2012; Enterlein et al., 2006; Watanabe et al., 2007), while VP40 and VP24 are responsible for assembly, budding and release of virion particles (Sobarzo et al., 2012; Enterlein et al., 2006). The NP encapsulates the genome and forms a complex with VP30, VP35 and L, which are required for both genomic replication and transcription of viral genes (Kondratowicz and Maury, 2012). The three other proteins, GP, VP40 and VP24 are membraned asso-

ciated proteins (Watanabe et al., 2007; Wilson et al., 2001) with GP playing an important role in inducing antibodies against Ebola virus. EBOV GP is a prime target for developing protective humoral immunity (Dhama et al., 2015). Reid et al. (2006) and Watanabe et al. (2007) opined that VP24 is very important for Ebola virus to evade the antiviral activities of interferons. Also, VP24, NP and VP35 are involved in the formation of nucleocapsid (Watanabe et al., 2007) and are essential for replication and encapsulation of the Ebola virus genome (Wilson et al., 2001). Ebola virus expresses two extra non-structural proteins from the GP gene referred to as soluble GP (sGP) being dominant and small soluble GP (ssGP) (Mire et al., 2012). Sullivan et al. (2003) described the two proteins as a soluble 60–70 kDa protein (sGP) and a full length 150–170 kDa protein (GP), leading to two fragments, GP1 and GP2, which are still covalently attached by disulfide bridges. GP1 has the receptor binding site while GP2 is anchored to the membrane. Other authors report these proteins as GP1 and GP2 (Martinez et al., 2011). Feldmann et al. (2003) reported that GP1–-GP2, which functions in receptor binding and fusion, is the target for the neutralizing host immune response. Wang et al. (2014) reported the central role of GP in the pathogenesis of Ebola virus and its role in vaccine discovery. Notwithstanding, it has been variously reported that vaccines for the protection from Ebola virus and other filovirus infections primarily target GP with exception of few targeting VP40 and/or NP (Mire et al., 2012; Sullivan et al., 2006; Martinez et al., 2011; Falzarano et al., 2011; Warfield et al., 2007a). A detailed review of the vaccine potential of Ebola virus proteins VP24, VP35, and VP40 can be found in Wilson et al. (2001). 3. Candidates vaccine Background research including vaccine discovery, proof of concept and preclinical trials in NHP, mice and guinea pig models are presented in Table 2. NHPs serve as the gold standard for animal models of Ebola infection and have been used to test candidate vaccines (Galvani et al., 2014). Promising vaccine candidates are listed in Table 3, though the WHO is currently coordinating phase I trials with two candidate vaccines: rVSV and rAd3 (WHO, 2014a,b,c; Butler, 2014). In the following subsections, candidates vaccines are described using several parameters such as replicating (replication competent) or non-replicating (replication deficient/incompetent), method of production i.e., whether conventional (irradiation, heat or formalin treatment) or molecular; whether vector based or not, the type of protection (pre exposure or post exposure prophylactic); dosage (singe, double or multiple), and valence (monovalent, bivalent or multivalent). Other information includes administration route, mode of action, phase of development and manufacturers’ name. 3.1. Replication competent vaccines Several promising vaccine candidates were developed from replication competent viral vectors. Prominent among these are recombinant vesicular stomatitis virus (VSV), recombinant human parainfluenza virus 3 (HPIV3), rabies and cytomegalovirus (CMV) (Table 3). According to Falzarano et al. (2011) replicating vaccines have several advantages over non-replicating ones including higher durability and long lasting immunity that typically require few modulations to confer protection. On the other hand vector based replication competent vaccines have several challenges such as the risk of reversal to pathogenicity, potential problems with pre-existing immunity and effects on patients with deficient or compromised immunity (Falzarano et al., 2011; Geisbert et al., 2010).

E.I. Ohimain / Virus Research 211 (2016) 174–185

3’ Leader NP VP35 VP40 Key NP= Nucleoprotein VP= Virus protein GP= Glycoprotein L= RNA dependent RNA polymerase

GP

VP30

VP24

177

L

5’ Trailer

Fig. 1. Simplified structure of Ebola virus genome.

Table 1 Functions of Ebola virus genes. Gene

Functions

References

NP VP35

Transcription and replication Transcription and replication RNA synthesis, type 1 interferon antagonist, virulence factor Virus assembly and budding Membrane associated and could possible play a role in viral entry Mediate viral entry into cell Transcription and replication Virus assembly and budding Membrane associated and could possible play a role in viral entry Type 1 interferon antagonist Transcription and replication

Enterlein et al. (2006), Sobarzo et al. (2012), Watanabe et al. (2007) Geisbert et al. (2010), Sobarzo et al. (2012), Enterlein et al. (2006) Watanabe et al. (2007), Enterlein et al. (2006), Leung et al. (2010), Trunschke et al. (2013) Kondratonicz and Magry (2012), Enterlein et al. Wilson et al. (2001), Feldmann et al. (2003) Enterlein et al. (2006), Sobarzo et al. (2012), Martinez et al. (2011) Enterlein et al. (2006), Sobarzo et al. (2012), Watanabe et al. (2007) Enterlein et al. (2006), Watanabe et al. (2007) Wilson et al. (2001), Feldmann et al. (2003) Geisbert et al. (2010), Kondratonicz and Magry, (2012) Watanabe et al. (2007), Sobarzo et al. (2012), Enterlein et al. (2006), Trunschke et al. (2013)

VP40 GP VP30 VP24

L

3.1.1. Recombinant vesicular stomatitis virus (rVSV) based vaccines Recombinant VSV Ebola virus vaccine was generated by using the wild-type VSV backbone and replacing the fusogenic VSV-G protein with the Ebola virus GP protein (Kanapathipillai et al., 2014). Another rVSV vaccine utilizes an attenuated form of VSV where the VSV N gene is shuffled from the first position (as in the wild-type) to the fourth. VSV belongs within the Vesiculovirus genus where the majority of these viruses infect domestic animals instead of humans, hence the challenge of pre-existing immunity will not arise. Other advantages of using VSV as an Ebola virus vaccine vector include growth to high titer (>109 pfu/ml) in vitro, propagation in almost all mammalian cells, induction of strong humoral and cellular immune responses, and the capacity to confer both mucosal and systemic immunity (Geisbert et al., 2010). VSV based vaccines can be administered through multiple routes such as intramuscular, oral and nasal delivery. The vaccine can confer both pre-exposure and post exposure protection (Geisbert et al., 2010; Falzarano et al., 2011; Marzi et al., 2013). There is a wealth of papers demonstrating the efficacy of the rVSV platform as an effective Ebola vaccine vector. Takada et al. (2003) demonstrated that rVSV based Ebola vaccine was effective and completely protected mice from a lethal Ebola virus challenge. Their data suggested that neutralizing antibody cocktails from passive prophylaxis and therapy of EVD can reduce the possibility of the emergence of antigenic variant in infected patients. Marzi et al. (2011) demonstrated an improved cross protection efficacy of heterologous rVSV based vaccine produced from Ebola virus VP40 of SUDV, protected against ZAIV in guinea pigs. In NHPs, cross protection was similarly demonstrated. Falzarano et al. (2011) demonstrated a single immunization with a monovalent rVSV vaccine expressing either the GP of ZAIV or TAFV when challenged with BDBV provided 75% cross protection in cynomolgus macaques. Though, complete protection requires incorporation of BDBV GP or a prime boost vaccine regimen (Mire et al., 2013; Dhama et al., 2015). This result suggests that monovalent rVSV Ebola vaccine can confer protection against newly emerging phylogenetically related species. The rVSV platform have been reported to be 100% protective in NHPs after mucosal immunization via intranasal route, which is considered the most likely scenario for a bioterrorism attack (Hoenen et al., 2012; Geisbert et al., 2008a). Furthermore, Geisbert and Feldmann (2011) shown that rVSV based vaccine expressing a single filovirus GP in place of VSV glycoprotein

(G) when injected in a single dose (blended) completely protected NHP against Marburg and 3 Ebola virus species. In a recent study, cynomolgus monkeys were vaccinated with a multivalent blended vaccine consisting of equal parts of the rVSVG GP vaccines for MARV, EBOV, and SUDV (Geisbert and Feldmann, 2011). At 4 weeks postvaccination, groups of the animals were challenged with MARV, ZAIV, SUDV, or TAFV (Table 2). Mire et al. (2013) also show that a single dose containing a blend of 3 rVSV vectors completely protected NHP against challenge with ZAIV, SUDV and TAFV and Marburg virus. Marzi et al. (2013) demonstrated the mechanism of vaccine protection using rVSV platform expressing ZAIV-GP against lethal Ebola virus in cynomolgus macaques, which suggests that antibodies played a significant role. In humans, rVSV was used to treat and manage a staff that was accidentally infected in a BSL 4 laboratory in Hamburg Germany (Hoenen and Feldmann, 2014). The safety of rVSV vector based Ebola virus vaccine is not in doubt. Mire et al. (2012) demonstrated that rVSV filovirus GP vaccine vector lack the usual neurovirulence associated with the wild type VSV parent vector. Geisbert et al. (2008b) demonstrated that rVSV vector expressing ZAIV GP is well tolerated and protected immunocompromised NHP during a lethal challenge. Studies have shown that rVSV vaccine has efficacy in eliciting both prophylactic and post exposure protection against Ebola virus (Feldmann et al., 2007; Galvani et al., 2014). At least two companies NewLink Genetics/Merck, and Profectus BioSciences Inc. (Table 3) have developed Ebola virus vaccines based on the rVSV platform, which are currently undergoing phase I testing. Canada’s rVSV Ebola vaccine development started in 2002 at Public Health Agency of Canada (PHAC) National Microbiology Laboratory, several investigational experiments were carried out from 2002 to 2012 but the commercial license was granted to New links Genetics in 2010. In 2013, 1500 vials of the vaccine was produced, out of which 500 doses were sent to WHO for clinical trials (PHAC, 2014). Preliminary results from the phase 1 clinical trials carried out at University Hospital of Geneva (HUG) indicated that the VSV–ZAIV is safe and immunogenic with some side effects including mild fever, and signs of arthralgia and vesiculation between the toes which is typical to a wild-type VSV infection seen in domestic animals. At high doses, other side effects include vaccine induced arthritis, dermatitis and vasculitis (Ledgerwood, 2015). Phase I trials have also began in USA, Canada, Germany and Gabon (HUG, 2014). Profectus BioSciences, Inc., have developed an attenuated rVSV based Filovirus vaccine called Vesiculovax for pre- and post- expo-

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Table 2 Experimental vaccines development. Vaccine type

Models

References

Research summary

Adenoviral vectors

Human

Ledgerwood et al. (2014)

NHP

Choi et al. (2015) Sullivan et al. (2000) Sullivan et al. (2003) Sullivan et al. (2006) Stanley et al. (2014)

Preliminary results of phase I clinical trial of ChAd3 based vaccines indicated safety and immunogenicity rAd based vaccine provide long term protection against Ebola virus Development of a preventive vaccine (DNA+Ad) for Ebola virus infection in primates. rAd based vaccine protected NHP against lethal Ebola virus challenge Low dose rAd vaccine protected NHP from lethal Ebola virus challenge Chimpanzee adenovirus vaccine boosted with MVA generates acute and durable protected macaques against lethal Ebola virus challenge. Multivalent Ebola vaccine based on Ad based platform Single CAdVax vaccine protected NHP against Ebola virus Chimpanzee Ad based vaccine protected guinea pigs against lethal Ebola virus challenge CAdVax bivalent vaccine protected mice against lethal Ebola virus challenge Enhanced protection of mice by Ad based vaccines Ad based vaccine protected mice against lethal Ebola virus challenge

Swenson et al. (2008) Pratt et al. (2010) Rodents Kobinger et al. (2006) Wang et al. (2006) Richardson et al. (2009) Patel et al. (2007) Vesicular stomatitis virus vectors

Human

Gunther et al. (2011) Geisbert and Feldmann (2011)

NHP

Geisbert et al. (2008a) Geisbert and Feldmann (2011) Mire et al. (2012) Falzarano et al. (2011) Marzi et al. (2013) Geisbert et al. (2008b) Feldmann et al. (2007) Mire et al. (2013) Rodents Wong et al. (2014) Marzi et al. (2011) Feldmann et al. (2007) Inactivated vaccines

Rodents Lupton et al. 1980 Rao et al. (2002) NHP

VEEV based replicons

Rodents Olinger et al. (2005) Pushko et al. (2001) Pushko et al. (2000) NHP

VLP

Rao et al. (2002)

Herbert et al. (2013)

Rodents Reynard et al. (2011) Martinez et al. (2011) Warfied et al. 2007a

NHP

Watanabe et al. (2004) Warfield et al. (2007b)

rVSV based vaccine was used to treat a lab staff accidentally exposed to Ebola vaccine in the lab rVSV based vaccine was used to treat a lab staff accidentally exposed to Ebola vaccine in the lab Post-exposure protection of NHPs by VSV based vaccines rVSV based vaccine protects against Ebola and Marburg viruses Safety of VSV based vaccines Cross protection of NHPs The mechanism of action of rVSV involves antibodies rVSV vaccines protected immunocompromised macaques Post exposure treatment of NHPs from EVD A blend of heterologous vaccine protected NHPs rVSV based vaccine protect guinea pigs and established the possibility of cross protection Improved cross protection efficacy in guinea pig Post exposure treatment of EVD Probably first attempt at producing Ebola vaccine tested in Guinea pigs Immune response of mice to Ebola virus after immunization with liposome encapsulated irradiated Ebola virus Immune response of monkeys to Ebola virus after immunization with liposome encapsulated irradiated Ebola virus Protective cytotoxic T-cell responses induced by VEEV replicons in mice Alpha virus replicon-based vaccine protected Ebola and Lassa virus in guinea pigs VEEV replicons based vaccine protected guinea pigs and mice from lethal Ebola virus challenge VEEV replicon based vaccine protected cynomolgus monkeys during lethal challenge with Ebola virus VLP based vaccine protected guinea pigs from lethal Ebola virus challenge Effects of Ebola mucin-like domain om anti-GP antibody responses induced by Ebola virus VLP in mice Filovirus-like particles produced in insect cells was immunogenic and confer protection in rodents VLP was produced from cDNA by reverse genetics Ebola virus VLP based vaccine protected monkeys during lethal challenge with Ebola virus

Humans Sarwar et al. 2014 Sarwar et al. (2015) Kibuuka et al. (2015) Martin et al. (2006) NHP Hensley et al. (2010)

DNA vaccines

Phase 1 trial of DNA vaccine indicated that the vaccine is safe and immunogenic Phase 1 trial of DNA vaccine indicated that the vaccine is safe and immunogenic Phase 1 trial of DNA vaccine indicated that the vaccine is safe and immunogenic Phase 1 trial of DNA vaccine indicated that the vaccine is safe and immunogenic Demonstrated cross protective DNA vaccine against lethal Ebola virus challenge in monkeys DNA vaccine against Filoviruses induced broad cytotoxic T-cells in mice and guinea pigs Rodents Shedlock et al. (2013) Phoolcharoen et al. (2011) Subunit vaccine protected mice against lethal Ebola virus challenge Mellquist-Riemenschneider et al. (2003) DNA and Baculovirus derived protein vaccines protected guinea pigs against lethal Ebola virus challenge

RABV

Blaney et al. (2013) NHP Rodents Blaney et al. (2011) Papaneri et al. (2012)

Bivalent vaccine confer immunity against Ebola virus and rabies in NHPs Bivalent vaccine confer immunity against Ebola virus and rabies in mice Bivalent vaccine confer immunity against Ebola virus and rabies in mice

CMV HPIV3

Rodents Tsuda et al. (2011) Bukreyev et al. (2007) NHP Meyer et al. (2015) NHP

A replicating CMV vaccine protected mice against Ebola virus challenge in mice Topical respiratory HPIV3 based vaccines protected monkeys against lethal Ebola challenge Aerosolized HPIV3/EboGP vaccine protected Rhesus macaques against lethal Ebola challenge HPIV3 based vaccines protected guinea pig against lethal Ebola challenge HPIV3 based vaccines protected guinea pigs against lethal Ebola challenge

Rodents Bukreyev et al. (2006) Bukreyev et al. (2009)

Abbreviations: HPIV3= Human para influenza virus type 3, RABV=Rabies virus; CMV=cytomegalovirus; r= recombinant; VEEV= Venezuelan Equine Encephalitis Virus; VLPs= Virus like particles; MVA=Modified Vaccinia Ankara.

Table 3 Promising first generation Ebola virus vaccine candidates. Vaccine A. Replicating rVSV–ZAIV-GP

Vesiculo vax Ebola RABV vaccine

rCMV

Mode of action

immunogen

Type of protection

Adm. Routea

Development status

Dose

Remarks

Newlink Genetics/ PHAC

Stimulates immune response to Ebola GP using rVSV Viral expression vector (VSV) Viral expression vector

GP

Post exposure / Prophylactic

IM or oral

Phase 3 ongoing

Single

GP

Post exposure / Prophylactic

IM or oral

Phase 1 ongoing

Replicating (Donated to WHO during outbreak) Trivalent vacccine

IM

Preclinical

Disseminating vaccine, long lasting Potential problems with pre-existing immunity

Profectus Biosciences Inc NIAID & Thomas Jefferson University Plymouth University

rHPIV3

B. Non replicating cAd3–EBOV

Ebola rAd5

Ebola GP Ad5

Glaxo SmithKline

Johnson & Johnson (Crucell/ Bavarian Nordic) University of Texas at Austin

Viral expression vector

GP, NP, VP40, VP35

Post exposure / Prophylactic

IN, IM, IP

Preclinical

single

recipients exhibited high EBOV-specific IgG, IgA, and neutralizing antibody titers

GP, NP, GP/NP

Preexposure/Prophylactic

IN, IM

Preclinical

Single

Viral expression vector (Adenovirus) Viral expression vector (Adenovirus) Viral expression vector (Adenovirus)

GP, NP, GP/NP

Prophylactic

IM

Phase 3

Single dose

GP, NP, GP/NP, GP/VP40

Preexposure/Prophylactic

Preclinical (Phase 1 ongoing)

Multiple dose

GP, NP, GP/NP

Prophylactic

IM

Phase 1

GP, NP, GP/NP, VP40/NP/GP GP, NP, GP/NP, VP40/NP

Pre-exposure / Prophylactic Pre-exposure / Prophylactic

SC

Preclinical

SC

Preclinical

Plasmid expression vector

GP, NP

Prophylactic

IM

Preclinical

Multiple

nano particles Viral expression vector (VEEV) MVA MVA, Immunostimulants

GP GP, NP, GP/NP, VP

IM

Phase 1 ongoing Preclinical

Multiple

GP GP

SC SC

VLPs FiloVax (rVLP protein)

SynCon

Integrated Bio-Therapeutics Inc Inovio pharmaceuticals Inc

EBOV GP ArV (replocon)

Novavax Inc Alpha vax Inc

GOVX-E301, GOVX-E302 EBOV Vaccinia

GeoVax Lab Bavarian Nordic

Live attenuated, GP

Preclinical Phase 1

Not affected by pre-existing Ad immunity

Multiple dose

E.I. Ohimain / Virus Research 211 (2016) 174–185

Manufacturer

DNA vaccines designed for multiple strains of Ebola

Single Protected immunecompromised population

Source: Bishop (2015); Yelle (2014); Feldman and Geisbert (2011); Norwegian Institute of Public Health (2014); Falzarano et al. (2011); Hoenen et al. (2012); Richardson et al. (2010); ADI (2014); Geisbert and Feldmann (2011). Abbreviations: HPIV3 = Human para influenza virus type 3; RABV = Rabies virus; CMV = cytomegalovirus; r = recombinant; VEEV = Venezuelan Equine Encephalitis Virus; VLPs = Virus like particles; MVA = Modified Vaccinia Ankara; IM = intramuscular; IN = intranasal; IP = intra peritoneal; SC = subcutaneous. a Vaccine administration routes.

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sure protection against Ebola virus. This vector uses an attenuated VSV backbone (Clarke et al., 2007; Clarke and Nasar, 2014) as compared to the NewLink backbone, which is wild-type. In addition the company is developing a trivalent filovirus vaccine against Ebola and Marburg viruses. A single dose of the vaccine has been demonstrated to confer protection to monkeys against lethal challenge with ZAIV (Mire et al., 2015). VesiculoVax is a trivalent vaccine that has been demonstrated to be able to protect NHPs from ZAIV, SUDV and MARV lethal challenge. The vaccine provided 100% protection of NHPs against 1000 times the lethal dose of the ZAIV. Profectus has secured a total of $27.9 million from US to develop the vaccine. 3.1.2. Rabies virus based vaccine platform Another platform showing promising results for Ebola virus vaccine is the Rabies virus (RABV). RABV is a non-segmented negative-strand RNA virus belonging to the family Rhabdoviridae. Ebola vaccines produced from RABV are replication competent i.e., it contains live attenuated virus. The vaccine is produced by replacing the RABV glycoprotein G gene with ZEBOV GP, which effectively reduced the neurovirulence of the virus and did not cause rabies after infection in mice (Hoenen et al., 2012; Blaney et al., 2011). Note that Rabies vaccine is currently being used for wildlife and domestic animals especially dogs in many parts of the world including Africa. RABV causes about 24,000 deaths in Africa yearly, hence a bivalent RABV/EBOV is apt (Blaney et al., 2011, 2013; Papaneri et al., 2012). The authors demonstrated the production of RABV/EBOV vaccines in 3 forms (1) replication competent RABV (2) replication incompetent RABV and (3) chemically inactivated EBOV expressing EBOV GP by reverse genetics system based on SAD BIG wildlife vaccine and tested them on mice (Blaney et al., 2011) and later on NHPs (Blaney et al., 2013). The results show in both mouse and NHP models show that the vaccine is protective, safe and immunogenic. Furthermore, the results show that IgG biased humoral responses and high levels of GP-specific antibodies are involved in conferring immunity in the tested animals. Based on the success of the preclinical studies, National Institute of Allergy and Infectious Disease (NIAID) collaborating with Thomas Jefferson University are pursuing the development of replication competent multivalent RABV/Ebola vaccine that could confer protection of NHPs and humans against RABV, ZEBOV, SEBOV and MARV. Papaneri et al. (2012) demonstrated that humoral immunity to GP could be induced by vaccination of mice with inactivated RABV/ EBOV GP even in the presence of pre-existing immunity to RABV. Clinical phase I trial is scheduled to commence mid 2015. It appears that the vaccine have now been licensed to Exxell Bio, USA for clinical trials and commercialization. 3.1.3. Recombinant cytomegalovirus (rCMV) based platforms Another promising platform for the development of EBOV vaccines is based on the replicating recombinant cytomegalovirus (rCMV) platform. Cytomegalovirus, which is a ␤-herpes virus (a DNA virus), is the largest virus known to infect humans. They initiate a predominantly asymptomatic infection in normal healthy individuals for life, low level persistent infection, which is benign and without evidence of viremia (Smith et al., 2013; Hoenen et al., 2012; Tsuda et al., 2011). Detailed review of CMV vaccines can be found in Smith et al. (2013) and Skenderi and Jonjic (2012). However, the major advantage of CMV based EBOV vaccine platform is that the virus is disseminating (Hoenen et al., 2012; Friedrich et al., 2012) i.e., once it has been established in a host, it can continue to replicate autonomously. This type of vaccine is quite relevant for the control of emerging and re-emerging zoonotic infections. It has been variously demonstrated that CMV has the ability to infect, re-infect and establish a persistent infection among diverse host regardless of pre-existing immunity (Tsuda et al., 2011). In a proof of concept study, Tsuda et al. (2011) demonstrated a replicat-

ing CMV based vaccine encoding a single Ebola virus NP of ZEBOV confers protection of mice against lethal ZEBOV challenge. 3.1.4. Human parainfluenza virus type 3 Another promising vector based platform for developing vaccines against the highly lethal Ebola virus is human parainfluenza virus type 3 (HPIV3). HPIV3 is a common pediatric respiratory pathogen. The virus is a member of Paramyxoviridae family. Like Ebola virus, it belongs to the order Mononegaviridae and are enveloped RNA viruses with a negative-sense RNA genome of 15–19 kb (Bukreyev et al., 2007, 2006; Yang et al., 2008). According to Yang et al. (2008), infection with HPIV3 induces both systemic and local respiratory tract immune responses to itself and when used as a vector to express foreign glycoproteins. A detailed review and research on paramyxovirus vectored Ebola vaccine can be found in Yang et al. (2008). Most studies on HPIV3 vaccine focused on intranasal vaccination, which could protect people from Ebola virus during bioterrorism attack, which could be transmitted through the nasal route. Bukreyev et al. (2006) demonstrated that paramyxovirus vectored vaccine can induce protective immunity against Ebola virus. They demonstrated the production of recombinant HPIV3 by modifying the virus to either express EBOV GP or together with NP. Results show that HPIV3 bearing Ebola GP is immunogenic and highly protective against Ebola virus challenge (Bukreyev et al., 2009) both in guinea pig (Bukreyev et al., 2006) and NHP (Bukreyev et al., 2007) models. The results of a follow up study in guinea pigs show that HPIV3 immune animals induced a very high level of EBOV antibodies and that the GP in the vector particles was not associated with increased replication in the respiratory tract (Yang et al., 2008). 3.2. Non-replicating (replication incompetent) vaccines Another group of promising Ebola virus vaccines different from earlier discussed are non-replicating vaccines. Details of nonreplicating Ebola vaccines can be found in several reviews including Hoenen et al. (2012), Friedrich et al. (2012), Falzarano et al. (2011), Richardson et al. (2010) and Mire and Geisbert (2014). Promising non-replicating Ebola virus vaccine include recombinant Adenoviruses, DNA vaccines and virus like particles (VLP), subunit vaccines, inactivated vaccines and replicons (Table 3). Replication deficient vaccines are generally safer because they do not contain live virus, hence the potential risk of reversion to virulence does not arise. 3.2.1. Adenovirus based vaccines Adenovirus (Ad) based vaccines are among the most studied non-replicating Ebola virus vaccine. Adenovirus presents a benign carrier system whose overall safety has been well demonstrated in various clinical trials (Greffex, 2014). Early research works of Yang et al. (1994, 1995, 1996) demonstrated the use of replication deficient adenovirus in gene therapy and showed the ability of these vectors to generate robust T and B cell responses to viral antigens. However, most of the first generation adenovirus vectors focused on the development of the vaccine based on human serotypes (AdHu5) (Kobinger et al., 2006; Greffex, 2014). Because a large number of human populations are exposed to this virus, pre-existing immunity reduced the effectiveness of AdHu 5 vaccines. Hence, second generation Adenovirus were developed from Chimpanzee Adenovirus particularly serotype 3 (cAd3). According to Hoenen and Feldmann (2014), the issue of pre-existing immunity has been effectively addressed by the use of various serotypes of Adenovirus such as the rare human serotypes Ad26 and Ad35, Chimpanzees Ad3, Ad7 and Ad62 and the simian Ad21. In mice model, Patel et al. (2007) demonstrated that AdH5 expressing Ad-ZGP delivered via oral and nasal vaccination pro-

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tected the animals against lethal Ebola virus challenge. Also, both T and B cell responses developed in the mice. Similarly, Richardson et al. (2011) demonstrated that a replication deficient rAdH5 based vaccine expressing ZEBOV GP protected guinea pigs and mice from Ebola virus. Sullivan et al. (2006) show how a single dose of Ad vector encoding modified Ebola GP protected NHPs from lethal Ebola virus challenge. Choi et al. (2015) also demonstrated protection of NHPs from lethal EBOV challenge with a single dose of respiratory Ad based vaccine. Immunization with DNA and/or replication—deficient rAd encoding Ebola GP and NP has been shown to confer specific protection immunity in NHPs (Sullivan et al., 2006). Ebola vaccine (cAd3 Ebola vaccine) is produced from Chimpanzee Adenovirus type 3 (cAd3) genome by deleting and replacing E1 gene with EBOV GP (Kanapathipillai et al., 2014). NIAID and Glaxo Smith Kline are developing candidate vaccine based on cAd3 platform. Phase I clinical trials have begun in the US, UK, Mali and Uganda. Preliminary results of phase I clinical studies show that cAd3-EBO vaccine induced EBOV specific glycoproteins antibodies that was in the range reported to be associated with vaccine induced protective immunity in challenge studies involving NHP (Ledgerwood et al., 2014). Of recent, there have been some improvements in the Adenovirus based vaccine platform. Kobinger et al., 2006) created a molecular clone of Chimpanzee Adenovirus pan 7 (AdC7) and used it to vaccinate mice and guinea pig in a lethal Ebola virus challenge experiment. Results show that both animals were protected completely while a robust T and B cell responses to ZEBOV were elicited even in the presence of pre-existing immunity. Richardson et al. (2009) demonstrated enhanced protection of mice against lethal Ebola virus challenge mediated by an improved Ad based vaccine. Results show that immune response and complete protection were achieved at a dose 100 times lower than that with previous generations of Adenovirus based Ebola virus vaccine. Research in the last 10 years are focused on the development of a single complex Adenovirus vector (CAdVax) candidate vaccines for protection against several strains of EBOV. Pratt et al. (2010) developed a bivalent vaccine that expressed ZEBOV and SEBOV GP in a single CAdVax and inoculated NHPs in a lethal EBOV challenge experiment via parenteral and aerosol routes. Resultsshow that the NHP were protected against both viruses. Wang et al. (2006) had earlier reported the development of a CAdVax based bivalent Ebola virus vaccine that induced immune responses against both Sudan and Zaire Ebola virus. Swenson et al. (2008) developed a polyvalent CAdVax that completely protected NHPs against most strains of Ebola and Marburg virus. Third generational Adenovirus based vaccines are emerging. Greffex (2014) announced the development of trivalent engineered Ebola-Marburg vaccine (GReEMTr) that expressed GP genes of ZEBOV, SEBOV and MARV with all Ad genes deleted. There are several advantages of this vaccine, which include targeted immune response, focused immune responses (antigen based on 2014 Guinea strain of ZEBOV), high immunogenicity and effectiveness at low doses, multi-delivery routes (intramuscular, intranasal, subcutaneous) and low immune interferences. Johnson and Johnson and Bavarian Nordic are also developing Ad26 and Ad35 vaccines. VaxArt is also developing and commercializing an Ebola virus vaccine VXA ZAIV-GP based on Ad5 platform. The candidate is a non-replication competent live oral monovalent Zaire Ebola virus vaccine. Challenged studies showed complete protection following single dose. Clinical phase I studies is expected to commence in 2015 (ASPR, 2015). The VaxArt vaccine can be administered orally as tablets. 3.2.2. DNA and subunit vaccines Vaccines using plasmid DNA have proven to be one of the most promising applications in the field of gene therapy (Geisbert et al.,

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2010). DNA vaccines are currently being developed for multiple pathogens such as hepatitis B virus, influenza virus, SARS and West Nile virus (Falzarano et al., 2011). Wahren and Liu (2014) reported recent developments and expression on the versatile use of DNA vaccines for protection against infectious diseases (HIV, Malaria, Ebola, HBV, Measles, Marburg, HSV, CMV, SARS, Influenza, dengue), cancers of the lungs, breast, prostate, bladder and other disease such as type I diabetes. The advantages of DNA vaccine platform are many. They include ease of production, and are readily scalable i.e., amenable to large scale production, non-infectious, it is not affected by host pre-existing immunity and have the ability to induce both cellular and humoral immunity (Falzarano et al., 2011; Geisbert et al., 2010). Results of preclinical demonstration of DNA and subunit vaccines for protection against Ebola virus have been reported. Shedlock et al. (2013) developed a synthetic polyvalent—filovirus DNA vaccine that protected mice and guinea pigs against lethal ZAIV, SUDV, and MARV virus challenge. The vaccination was reported to be highly potent, which elicited robust neutralizing antibodies that completely protected the animals against Ebola virus. The vaccine induced a cytotoxic T lymphocytes of great magnitude. Hensley et al. (2010) demonstrated that a DNA prime/rAd5 vaccine expressing ZAIV and SUDV GP can induce cross protection in cynomolgus macaques against all three lethal Ebola viruses ZAIV, SUDV, TAFV. Vaccinated animals developed robust antigen-specific humoral and cellular immune responses against the GP from ZAIV and cellular immunity against BDBV. Phoolcharoen et al. (2011) demonstrated how a non-replicating subunit vaccine protected mice against lethal Ebola virus challenge. Mellquist-Riemenschneider et al. (2003) vaccinated guinea pigs with Baculovirus-derived GP alone and in combination with a DNA prime Baculovirus protein boost regimen, developed antibody responses but incomplete protection was achieved in Ebola virus challenge, whereas this same procedure completely protected the animals from Marburg virus. Numerous studies have demonstrated the safety and immunogenicity of DNA-based Ebola virus vaccines. In a phase I clinical trial, Martin et al. (2006) demonstrated that a three plasmid DNA vaccine encoding for envelope GP from ZAIV and SUDV and NP was well tolerated with no adverse effects or coagulation abnormalities. Other authors have similarly demonstrated the safety and immunogenicity of polyvalent DNA vaccines encoding Ebola virus and Marburg virus GP in a phase I clinical trials (Sarwar et al., 2015; Kibuuka et al., 2015). The results show that the vaccines were well tolerated and elicited antigen specific cellular and humoral immunity. Inovio Pharmaceuticals Ltd., is developing and commercializing a DNA based Ebola vaccine that was designed using the company’s syncon technology to provide broad protective antibody and Tcell responses against multiple strains of Ebola virus. The vaccine is currently at phase I clinical trials. The vaccine could be administered via electroporation. Protein Science is also developing and commercializing an Ebola glycoprotein vaccine based on Baculovirus expressed recombinant protein. Clinical trial is expected to begin from first quarter of 2015. 3.2.3. Virus like particles Another platform for developing Ebola virus based vaccines is through virus like particles (VLP). Enveloped VLP can be generated in a mammalian expression system by introduction of the gene coding for VP40 Matrix protein (Warfield et al., 2007a). Martinez et al. (2011) and Warfield et al. (2007b) reported that VP40 induces the production of VLPs that are biochemically and morphological similar to Ebola virus. Using reverse genetics, Watanabe et al. (2004) generated Ebola VLP from cDNA that was indistinguishable from that of intact/authentic Ebola virus. Richardson et al. (2010) reported that VP40 or along with GP can induce the for-

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mation of VLP. Co-expression of other proteins such as GP, VP24, VP40 and NP alone or in combination will result in the generation of Ebola VLPs (eVLP), containing these viral antigens (Warfield et al., 2007b). When GP is co-expressed with VP 40, the GP, which becomes incorporated into the eVLP, could mediate entry into cells (Martinez et al., 2011). Generally, VLP lacks NP, VP30, VP35 and L proteins. Filovirus VLP vaccines have used particles containing 2–3 proteins (VP40, GP, or NP) (Warfield and Aman, 2011). The advantages of VLP based vaccine platforms are many. Because they lack the RNA genome, they are not infectious i.e., non-replicating, hence, there are no concerns with respect to reversion of virulence, which is a major safety concern in replicating vaccines. Also, VLPs are effective even in the presence of host pre-existing immunity, which is a major drawback of vector based vaccine platforms (Richardson et al., 2010). A detail review on advances in VLP vaccines against filoviruses can be found in Warfield and Aman (2011). ADI (2014) reported that VLP have the advantages of rapid production in large quantities and can generate robust innate, humoral and cellular immunity in animals and humans, and a single dose can be effective against ZAIV, SUDV and MARV. However, Ebola VLP vaccines have shown promising but sometimes contradictory results in rodents and NHP models. Warfield et al. (2007a) demonstrated the production of eVLP from insect cells and were used to vaccinate mice, which generated antibody and cellular responses equivalent to those vaccinated with mammalian 293T cell derived eVLP and the mice were protected from lethal Ebola virus challenge. Similarly, Reynard et al. (2011) demonstrated that VLPs containing Kunji virus replicon-based vaccines expressing Ebola virus GP protected guinea pig against lethal Ebola virus challenge. In a mouse model, Martinez et al. (2011) demonstrated that Ebola virus mucin-like domain can increase anti-GP titres induced by eVLP. Unsuccessful or partial protection of guinea pigs by VLP has been reported. In NHP models, Warfield et al. (2007b) demonstrated that eVLP-based vaccine protected monkeys against lethal Ebola virus challenge. NovavaxInc is developing and commercializing two products, a Baculovirus-derived Ebola GP nano particles vaccine and matrix (sapronin) adjuvant. The vaccine was based on the Guinea strain isolated during the ongoing 2014 epidemics in West Africa. Preclinical studies show that the vaccine protected 100% of mouse in a lethal challenge, and demonstrated high immunogenicity in mice, rabbits and baboons (ASPR, 2015). Novavax Ebola virus vaccine plus adjuvant induced high neutralizing antibody levels that cross neutralized the more virulent Ebola virus 1976 strain with antibody titers well within ranges reported to protect against Ebola virus infection (Smith, 2014). Phase I clinical trials commenced in December 2014. Smith (2014) reported that the Novavax recombinant EBOV GP vaccine was correctly folded and efficiently produced with a scalable technology where the purified nanoparticles were highly immunogenic with Matrix-M adjuvant in inducing crossneutralizing antibodies.

of Ebola virus. Pushko et al. (2000) demonstrated that rRNA replicons derived from attenuated VEEV protected guinea pigs and mice from lethal Ebola virus challenge. In mouse model, Olinger et al. (2005) demonstrated the protective cytotoxic T-cell responses were induced by VEEV replicons expressing Ebola virus protein. Pushko et al. (2001) demonstrated VEEV replicon protected guinea pigs against Lassa and Ebola virus infections. Herbert et al. (2013) demonstrated that VEEV replicons particles vaccine completely protected cynomolgus macaques from intramuscular and aerosol challenge with SEBOV. Alpha Vax Inc., is developing and commercializing Ebola vaccine based on VEEV replicon. 3.2.5. Vaccinia based ebola vaccines Vaccinia virus (VACV) is a large, complex and enveloped virus belonging to the smallpox family. Vaccinia vaccine, which was initially developed and used to eradicate smallpox, has been more used extensively for human immunization than any other vaccine (Jacobs et al., 2009). A review on vaccinia vaccines can be found in Jacobs et al. (2009), Price et al. (2013), Gibert (2013). Current application of VACV is based on the modified Vaccinia virus Ankara (MVA)-T RNA polymerase promoter (Geisbert and Jahrling, 2003). Recombinant vaccinia viruses are constructed by exploiting homologous DNA recombination in vaccinia virus infected cells where the foreign gene is inserted (Geisbert et al., 2010). MVA is an attenuated, replication deficient vaccinia virus (Price et al., 2013; Gibert, 2013) that is highly immunogenic and hence developed to combat several diseases including cancer, influenza, tuberculosis, malaria, HIV and is now being considered for Ebola virus. Price et al. (2013) listed the many benefits of MVA to include enhanced safety due to the replication incompetency of the vaccines, hence no risk of reversion of virulence, MVA efficiently express viral and recombinant genes and rapid elicitation of immune responses. Though, there are not much report on preclinical demonstration of the efficacy of MVA on Ebola virus, companies have started development of the vaccine. In a phase 1a and 2a clinical trials, Kreijtz et al. (2014) demonstrated MVA based vaccine against influenza virus, H5N1. The vaccine is safe and immunogenic. At least, two companies have started developing and commercializing MVA based Ebola virus vaccines. Bavarian Nordic of Denmark initiated its phase I clinical trial with MVA-BN® Filo and the AdVac® technology from Crucell Holland B.V., one of the Janssen Pharmaceutical Companies of Johnson & Johnson. The vaccination with MVA is benefited by a heterologous prime-boost vaccination regiment and not given alone. Large scale clinical trial is expected to commence in April 2015 (Bavarian Nordic, 2015). Geo Vax Labs Inc., has announced the development and commercialization of recombinant MVA vaccines designed to produce non- infectious VLP Ebola virus GP. The vaccine GOVx-E301 was designed as a single dose vaccine against the current 2014 ZAIV, while GOVX-E302 vaccine was designed to protect against the 3 known lethal Ebola virus species (Geo-Vax Labs Inc., 2014).

3.2.4. Venezuelan Equine Encephalitis virus replicon vaccine Another vector-based vaccine platform that has proved effective against EBOV is replicons produced from Venezuelan Equine Encephalitis Virus (VEEV). VEEV is an alpha virus whose genome consists of single stranded, positive-sense RNA divided into 2 open reading frames encoding for structural and nonstructural proteins (Geisbert et al., 2010). The VEEV structural proteins can be modified by adding genes of interest such as GP or NP of EBOV resulting in a replicon (Falzarano et al., 2011). RNA in these replicon containing VLP are used as vaccines (Hoenen et al., 2012). Olinger et al. (2012) reported that VEE replicon expressing Ebola virus GP, NP, VP 24, VP30, VP35 and VP40 protected against Zaire Ebola virus. In mice and NHP which can be administered by various routes via a single dose is efficacious against multiple strains

3.2.6. Inactivated vaccines Conventional inactivated vaccines have been produced from Ebola virus by heat, formalin or gamma irradiation (Richardson et al., 2010; Hoenen et al., 2012). Lupton et al. (1980) vaccine demonstrated that inactivated Ebola virus vaccine was effective in protecting guinea pigs in a lethal Ebola virus challenge. To the best of our knowledge, this is probably the first demonstration of inactivated vaccines against the Ebola virus. But because of the potential risk of reversion to virulence, conventional inactivated vaccines are not often supported against Ebola virus. Rao et al. (2002) demonstrated the induction of immune responses in mice and monkeys to Ebola virus challenge after immunization with lipososome-encapsulated irradiated Ebola virus, mice were protected but monkeys succumbed to the disease. Other reports

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suggest that conventional viruses are not effective against the Ebola virus. 3.2.7. Other vaccines Apart from the vaccines reviewed in this publication, there are several others that are at various stages of development from proof of concept, preclinical and some have started clinical trials with little or no reporting. Vaccines produced in Russia for example are scarcely reported. Some other promising vaccine candidates include DPX-Ebola (Immunovaccine Inc.), Baculovirus Expression Vector System (Protein Sciences Corporation), Nasal vaccines (University of Texas at Austin), TK-05 (Sihuan Pharmaceuticals). Others include recombinant filovirus vaccine (University of Hawaii) and recombinant Ebola vaccine with VP 30 or VP35. 4. Conclusion Ebola virus disease is one of the major threats to global peace and security. Ebola virus is a zoonotic virus causing hemorrhagic fevers in humans and primates. Since 1976 when the virus was first discovered, over 20 outbreaks have occurred mostly confined to rural areas in East and Central Africa. But the 2014 outbreak, which started in three West Africa countries namely Guinea, Sierra Leone and Liberia, took a different pattern. It is the first time the virus would emerge in urban centers and spread beyond Africa into Europe and USA. Thus far, over 22,000 cases have been reported with nearly 50% mortality in nearly one year. Zaire Ebola virus, which is the causative agent of the current outbreak, has fatality rates of up to 90%. Though, the current outbreak recorded an improved mortality rates, but the magnitude of the outbreak is greater than all the other outbreaks combined. The spread of Ebola virus in the current outbreak beyond Africa and the high fatality rates, has spurred research into the development of preventive vaccines and therapeutics. Vaccines appear to be at least a step faster than therapeutics. Conventional inactivated vaccines produced from Ebola virus by heat, formalin or gamma irradiation appears to be ineffective and there is also the risk of reversion to virulence. Hence, novel vaccines production techniques were adopted leading to the production of candidate vaccines that have been demonstrated to be effective in preclinical trials using small animal and NHP models. Some of the promising vaccines have undergone phase 1 clinical trials, which demonstrated their safety and immunogenicity. Many of the candidate vaccines are vector based such as VSV, RABV, Ad3, MVA, CMV, HPIV3 and VEEV. Other platforms include VLP, DNA and subunit vaccines. WHO is currently carrying out/coordinating clinical trials of Ad and VSV based vaccines. Acknowledgement The author wishes to thank Sylvester C. Izah of the Niger Delta University for the editorial work and Dr. Beth Middleton of USGS for proofreading the manuscript. The author also wishes to thank the two anonymous reviewers for their useful comments and suggestions. The author wishes to dedicate this manuscript to all the field staff of international NGOs (listed here: http://www.cidi.org/ebolangos/#.VPTjsC5vTFU assessed 2 March 2015) who risked their lives while combating Ebola virus disease in West Africa, funding agencies (Bill and Melinda Gates Foundation, Facebook, Aliko Dangote Foundation, Tony Elumelu Foundation), United Nations and some her agencies such as WHO and UNICEF, banking institutions (AfDB, World Bank) and the government of nations that responded to the Ebola Crisis and c countries that funded research for the development of drugs and vaccines (USA, Canada, Japan, China, Cuba, UK, Spain).

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