REVIEW
review
Human Vaccines 6:6, 439-449; June 2010; © 2010 Landes Bioscience
Recent advances in Ebolavirus vaccine development Jason S. Richardson,1 Joseph D. Dekker,2 Maria A. Croyle2 and Gary P. Kobinger1,3,* Special Pathogens Program; National Microbiology Laboratory; Public Health Agency of Canada; Winnipeg, MB Canada; 2Institute of Cellular and Molecular Biology; The University of Texas at Austin; Austin, TX USA; and 3Department of Medical Microbiology; University of Manitoba; Winnipeg, MB Canada
1
Key words: ebolavirus, virus, vaccines, route, formulations Abbreviations: EBOV, Ebolavirus; ICEBOV, Cote d’Ivoire EBOV; REBOV, Reston EBOV; BEBOV, Bundibugyo EBOV; SEBOV, Sudan EBOV; ZEBOV, Zaire EBOV; NP, nucleoprotein; VP,viral protein; GP, glycoprotein; L, RNA-dependent RNA polymerase; I.M., intramuscular; I.N., intranasal; OR, oral; NHPs, non-human primates; I.P., intraperitoneal; VSV, Vesicular Stomatitis Virus; HPIV-3, Human parainfluenza virus type 3; VLP, virus-like particle
Ebolavirus is a highly infectious pathogen with a case fatality rate as high as 90%. Currently there is a lack of licensed Ebolavirus vaccines as well as pre- and post-exposure treatments. Recent increases in the frequency of natural human Ebolavirus infections and its potential use as a bioterrorism agent makes vaccine development a priority for many nations. Significant progress has been made in understanding the pathogenesis of Ebolavirus infection and several promising vaccine candidates were shown to be successful in protecting NHPs against lethal infection. These include replication-deficient adenovirus vectors, replicationcompetent VSV, HPIV-3 vectors and virus-like particle preparations. Recent advances in the generation of effective post-exposure immunization strategies highlight the possibility of developing a single dose vaccine that will confer full protection in humans following Ebolavirus exposure. Postexposure protection is particularly important in outbreak and biodefense settings, as well as clinical and laboratory settings in the case of accidental exposure.
Introduction Filoviruses are enveloped, non-segmented, negative-strand RNA molecules taxonomically assigned within the order Mononegavirales1 and family Filoviridae.2-12 They are further divided into the genera Marburgvirus (MARV) and Ebolavirus (EBOV).13 Five species of EBOV have been identified: Cote d’Ivoire EBOV (ICEBOV),14 Reston EBOV (REBOV),15 Bundibugyo EBOV (BEBOV),16 Sudan EBOV (SEBOV)17 and Zaire EBOV (ZEBOV),18 whereas, the Marburgvirus genus contains only one species, Lake Victoria marburgvirus (MARV).12,19,20
*Correspondence to: Gary P. Kobinger; Email:
[email protected] Submitted: 06/17/09; Revised: 12/23/09; Accepted: 01/04/10 Previously published online: www.landesbioscience.com/journals/vaccines/article/11097
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Natural EBOV infections are primarily localized to the humid rain forests of Central and western Africa as well as the Philippines.21,22 While the precise mechanism of natural virus transmission to humans and non-human primates (NHPs) remains elusive, there are some indications that bats may constitute the natural reservoir and primary source of infection.23-25 To date, there have been approximately 1,900 confirmed cases of human EBOV infection with over 1,340 deaths. The emergence and re-emergence of ZEBOV is responsible for the most frequent and lethal outbreaks. In 1976, the first reported ZEBOV outbreak occurred in the Democratic Republic of the Congo with an 88% fatality rate.18 Subsequent outbreaks of ZEBOV have been sporadic with high mortality rates, ranging from 57–90% (Table 1). SEBOV is the second most prevalent Ebolavirus species, with four reported outbreaks. Mortality rates associated with SEBOV infections range from 41 to 65% (Table 1).17 In 2007, an outbreak of haemorrhagic fever in the Bundibugyo District of western Uganda was shown to be caused by a newly isolated EBOV classified as BEBOV.26 Although REBOV and ICEBOV have been found to be pathogenic in NHPs, there has only been one reported non-fatal human case of ICEBOV.14,27,28 There are no documented human fatalities due to REBOV infection.15,29,30 EBOV contains a linear negative-sense RNA genome that is approximately 19 kb in length.10 The EBOV genome contains seven genes that sequentially encode a nucleoprotein (NP), two viral proteins (VP35 and VP40), a glycoprotein (GP), two additional viral proteins (VP30 and VP24) and a RNA-dependent RNA polymerase (L) (Fig. 1A). NP is the major ribonucleoprotein that aggregates with the minor ribonucleoprotein, VP30, and together they form a complex with VP35 and L. The interaction between this complex and viral genomic RNA creates the nucleocapsid.31 Transcription and replication of the RNA genome is accomplished by the L and VP35 proteins.12,32 VP40 is the most abundant protein and it is critical for virion assembly.31 Oligomers of VP40 bind singlestranded RNA and along with VP24, form the matrix protein.31,33,34 VP24 is involved in virion assembly and is a potent inhibitor of the type I interferon response.33,35 The GP gene encodes the glycoprotein (GP) consisting of two subunits, GP1
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Table 1. Total number of cases and fatalities associated with EBOV outbreaks over time* Bundibugyo # of cases
Cote d’Ivoire
Fatal (%)
# of Cases
Sudan
Fatal (%)
Zaire
# of cases
Fatal (%)
# of cases
Fatal (%)
284
53%
318
88%
1
100%
34
65%
1994
52
60%
1995
315
81%
1996
37 60
57% 74%
122
79%
2002
143
89%
2003
35
83%
12
75%
1976 1977 1979
2001
425
2004
17
2005 2007
1 149
0%
25%
2008–2009 *
53%
41% 264
71%
32
47%
Data taken from references 182–193.
Figure 1. Schematic representation of (A) organization of filovirus genomic RNA (ZEBOV depicted); l, Leader sequence; NP, nucleoprotein gene; VP, viral protein gene; GP, glycoprotein gene; L, RNA-dependent RNA polymerase gene; ir, intergenic region and (B) filovirus particle; NP, nucleoprotein; VP, viral protein; GP, glycoprotein; L, RNA-dependent RNA polymerase.
and GP2 . A domain on the N-terminus of GP1 contains a cellsurface receptor-binding domain.36-39 Antibodies specific to this domain can prevent cell entry40 (Fig. 2). It also contains an immunosuppressive domain, located at the N-terminus of the transmembrane domain, which may be released upon proteolysis of spike proteins during infection.41-43 GP2 is involved in fusion of the EBOV envelope with the host cell membrane following GP1 binding to a cell-surface receptor. Transferrin and DC-sign have both been proposed as cellular receptor(s) involved in mediating filovirus entry through binding and internalization, although their real contribution to virus entry remains controversial.44 GP1 and GP2 are capable of forming GP1,2 heterodimers. GP1,2 heterodimers are classified as type I transmembrane proteins. The heterodimers can be N- and O-glycosylated,45-48 acylated and phosphorylated.12,43 GP1,2 heterodimers form trimers, and collectively they make up the
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EBOV spike protein.49-51 The spike proteins contain a variable central region for which monoclonal antibodies against EBOV and MARV have been generated.52-54 EBOV infection causes hemorrhagic fever in humans. The EBOV incubation period ranges from 2–14 days and death typically occurs between day 6 and 16, although late mortality at 28 days post-onset of the first symptoms was documented.12 While the sequence of infection is not completely understood, pathology studies in NHPs have shown that dendritic cells, tissue macrophages and monocytes are the primary targets of filovirus replication, in addition to endothelial cells, fibroblast, hepatocytes and adrenal cortical cells.55-66 The release of chemokines from infected cells upregulates monocytes and macrophage recruitment.57,67 The recruitment of these additional cells promotes viral replication and transport of virus to the lymph nodes and spleen.57,67 Although EBOV does not infect lymphocytes, there is a rapid loss
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Figure 2. Schematic view of the process associated with EBOV entry, lifecycle and a model of virion budding and release. Symbols are as follows: NP shaded circles, VP30 filled circles, VP35 diamond, L triangle, VP40 filled rectangle, GP three shaded circles. Attachment of the virion to an unidentified host surface receptor results in receptor-mediated endocytosis or direct fusion with the host cell membrane. The virion is acidified within the endocytic vesicle leading to the uncoating of the nucleocapsid and release of the viral RNA genome into the cytoplasm of the host cell. Polyadenylated monocistronic mRNA is synthesized from the negative-sense genomic RNA template by the replicase-transcriptase holoenzyme. Translation of the viral mRNA genome yields the filoviral structural proteins. Viral replication of the positive-sense antigenome serves as a template for the generation of the negative-sense progeny genomes. Prolonged replication produces excessive amounts of viral proteins, which facilitates transition from transcription/translation to replication within host cells. The concentration of the nucleoprotein NP is the primary trigger that induces this transition between mRNA transcription/translation and genomic replication. In vitro studies have shown VP40 contact multivesicular bodies (MVB), dimerize and form octomeric rings inducing a conformational change which results in oligomerization and exposure of late budding motifs. These motifs recruit Nedd4 and ESCRT-1, ESCRT-1 then recruits ESCRT-II and ESCRT III. Then the GP1,2 transmembrane domain may direct GP1,2 from the trans-Golgi network to the multivesicular bodies containing VP40. The ESCRT complex dissociates from the multivesicular bodies by Vps4 ATPase. NP, VP30, filoviral RNA, along with the replicase/transcriptase holoenzyme (L-VP35) are directed to the multivesicular bodies. Binding of NP to progeny genomic RNA with VP35, VP30 and L result in the formation of ribonucleocapsids. Following assembly, virions are released from the host cell by budding.
of these cells during infection through apoptosis.57,68-71 Late stage filovirus infection leads to a inefficient immune response with pro-inflammatory mediators such as cytokines and chemokines failing to activate T- and B-cell responses and instead inducing haemorrhage, shock and multiple organ failure.12,60,67,72-81 EBOV is classified as a biosaftey level 4 pathogen by the World Health Organization due to its lethality of infection, ease of human-to-human transmission, and lack of approved therapies.12,82 In the United States, filoviruses are designated Category A agents by the Centers for Disease Control and as such are considered potential agents of bioterrorism, posing a risk for largescale dissemination.12
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Therapeutic Options for EBOV Infections Approximately 4–10 days following initial EBOV infection, patients rapidly develop a fever and other symptoms (headache, weakness, sore throat), eventually progressing to a maculopapular rash and signs of impaired coagulation. Unfortunately, clinical EBOV cases are often misdiagnosed since the incubation time and flu-like symptoms are common to several other infectious diseases. Cases are frequently identified only after failure to respond to anti-malarial and/or antibiotic regimens, thereby increasing exposure to the general public and healthcare workers. Even if EBOV is identified early during infection, effective
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anti-viral therapies are not currently available. Instead, treatments are primarily supportive and include hydration, blood volume maintenance, pain management, as well as interferon regimens when available.83 Anticoagulation treatments using recombinant nematode anticoagulant protein c2 (rNAPc2) have had some success for post-exposure protection of NHPs. rNAPc2 inhibits the f VIIa/tissue factor complex that when overexpressed is likely involved in triggering coagulation abnormalities and thrombosisrelated organ failure in EBOV infected primates. 33% of EBOV infected NHPs administered rNAPc2 survived challenge and prolonged survival times compared to the controls were reported for treated NHPs that did not survive. Attenuated coagulation and proinflammatory responses were cited as some of the probable factors leading to protection.84 Similar results were reported using recombinant human activated protein C (rhAPC), which is involved in regulation of blood coagulation and inflammation. In primates, ZEBOV hemorrhagic fever results in rapid decrease of plasma levels of protein C. NHPs administered rhAPC following challenge with ZEBOV resulted in 18% protection and a prolonged mean time to death of non-survivors as compared to control animals.85 In addition to these therapeutic treatments, the development of effective prophylaxis to prevent EBOV infection would be beneficial for communities, healthcare workers, and NHPs in filovirus-endemic regions, as well as against accidental laboratory exposure.43 Conventional Vaccines Conventional inactivated vaccines have been generated through inactivation of EBOV by heat, formalin, or γ-irradiation. Several conventional vaccine candidates were not effective at stimulating protective immune responses.86-90 Protection in NHPs against EBOV was not observed following vaccination with inactivated virus, with or without liposome or adjuvant, despite altering the route of administration (subcutaneous (S.C.) vs. intramuscular (I.M.) injection) or adjusting the timing of booster injections.91 There is a significant risk of reversion associated with liveattenuated filovirus vaccine candidates making safety a major concern. This is supported by studies showing the retention of virulence of mouse-adapted and guinea pig-adapted EBOV in NHPs.43,92-95 Alternatively, genetic engineering may provide a safe yet immunogenic attenuated EBOV vaccine candidate. Recently, mice vaccinated with two doses of a replication-deficient EBOV lacking the VP30 gene and protein product protected mice and guinea pigs from lethal challenge.96 Sub-Unit Vaccines (Non-Viral) EBOV genes inserted into a DNA plasmid can be injected directly into a patient’s muscle, where expression of the antigen can elicit an immune response to the corresponding virus particle. The use of DNA vaccines can be advantageous as they can lead to the generation of antibody and cytotoxic T lymphocytes.97 Additionally, they are easily manufactured, cost effective and are stable for storage and shipping at ambient temperatures.98 Several methods of DNA vaccine delivery have been used including
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direct administration into tissue via syringe, gene gun delivery of DNA, or electroporation of muscle tissue following injection of DNA. DNA vaccines expressing EBOV antigens GP, NP, VP40 or VP35 alone or in combination have been evaluated in mice, guinea pigs and NHPs.99-101 Full protection was reported in mice and later in guinea pigs with optimized strategies.99-101 In studies using EBOV DNA vaccines, consistently low survival rates have been documented for NHPs.102,103 Amino acid sequence computer analysis has identified several candidate peptides that may be useful in developing peptide-based vaccines using known peptide-immune cell interactions. Several ZEBOV-specific mouse cytotoxic T lymphocyte epitopes have been identified from the amino acid sequence of the EBOV proteins NP, VP35, VP40, GP, VP30 and VP24. Despite the presence of neutralizing antibodies and cytotoxic T lymphocytes in vaccinated NHPs, these animals did not survive a lethal challenge with EBOV.104 Virus-like Particles (VLPs) as a Candidate Vaccine VLP vaccines, unlike viral vaccines inactivated by heat, chemical or γ-irradiation, can present filoviral antigens in a presumably native form. EBOV-like particles have been examined as potential vaccine candidates and can be generated by the expression of VP40 alone or along with GP.105-115 While these surface proteins assemble much like infectious EBOV particles, VLPs lack NP, VP35, VP30, VP24 and L proteins. The EBOV RNA genome is also absent and the particle is therefore non-infectious. The biosafety concerns regarding reversion of attenuated filovirus vaccines are thereby eliminated with VLPs. Frequently, vectorbased vaccines expressing EBOV epitopes can be subject to host pre-existing immunity to the vector backbone, thereby inhibiting vaccine efficacy by preventing an immune response to the antigen. VLP-based vaccine platform have an additional advantage as they can circumvent these pre-existing immunity issues. Mice vaccinated with VLPs expressing ZEBOV VP40 and ZEBOV GP followed by either two booster injections, or one booster with QS-21 adjuvant, resulted in complete protection from a challenge with a lethal dose of mouse-adapted ZEBOV.114,116 In these experiments, this vaccine generated CD4 + and CD8 + T cells specific to GP and VP40 peptides, mouse IgG responses, and B cell activation with no toxic cytokine response.114,116 Neutralizing antibodies were also induced in a dose-dependent manner.114 Additionally, the concentration of natural killer (NK) cells increased in VLP vaccinated mice. Challenge of VLP vaccinated mice deficient or depleted of NK cells resulted in incomplete protection, whereas wild-type mice treated with VLPs were fully protected.117 Full protection and high titers of antibodies to ZEBOV were detected in ZEBOVchallenged guinea pigs vaccinated with VLPs in combination with RIBI adjuvant.112 Full protection was observed, without clinical or laboratory signs of EBOV infection, in NHPs vaccinated with VLPs containing EBOV GP, NP and VP40 following lethal challenge.118 EBOV specific antibody titers were detected from the serum of VLP-vaccinated NHPs and CD44 + T cells responded with vigorous production of TNFα after EBOV-peptide re-stimulation.118
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Vector-Based Vaccines Viruses can be used as vaccine vectors when genes encoding antigens of EBOV are inserted and expressed from the viral carrier. Viral vectors can be replication competent or defective. While replication competent vectors generally elicit strong and long-lasting immune responses following immunization, these platforms may not be recommended for use in immunocompromised individuals. Defective viral vectors, while potentially safer, may require multiple doses to achieve optimal immunity. Vaccinia virus-based vaccines. Vaccines based on recombinant vaccinia virus expressing ZEBOV GP, VP24, VP35 and VP40 were tested in the guinea pig model. Following lethal ZEBOV challenge, a recombinant vaccinia virus expressing ZEBOV GP afforded incomplete protection in guinea pigs. None of the other recombinant vaccinia-based vaccines expressing the other ZEBOV antigens protected the guinea pigs from succumbing to the infection.119 Although neutralizing antibodies against ZEBOV were detected, NHPs administered recombinant vaccinia virus expressing ZEBOV GP did not protect the animals against lethal homologous challenge.91 VEE virus-like replicon particles. Venezuelan equine encephalitis (VEE) virus has a positive-sense RNA genome. The VEE virus structural genes can be replaced by EBOV NP or GP, generating replicons that are single cycle and propagation deficient. VEE replicons expressing ZEBOV GP (VEE-GP), NP (VEE-NP), or a combination of GP + NP (VEE-GP + NP) were shown to be fully protective in mice challenged with ZEBOV.120 Strain 13 guinea pigs vaccinated with VEE-GP followed by homologous ZEBOV challenge resulted in 100% protection.120 When this experiment was repeated in strain 2 guinea pigs the results were not as definitive as only 60% survival was reported, despite high antibody titers against ZEBOV GP.120 Vaccination with VEE-GP, VEE-NP or VEE-GP + NP followed by two booster injections resulted in no protection of NHPs challenged with ZEBOV.91 Adenovirus-Based Vaccines. Initially human adenovirus serotype-5 (AdHu5) was mostly used as the adenovirus-based vaccine prototype. AdHu5 is well characterized; it can cause mild respiratory disease, gastroenteritis and conjunctivitis in humans. The recombinant AdHu5 backbone contains deletions in the intermediate early E1 gene. This deletion prevents virus replication and provides a site for introduction of foreign DNA such as viral antigens. Additional deletions to the E3 and E4 regions of AdHu5 have increased the carrying capacity of this vector to approximately 8 kb. Recombinant AdHu5 vectors are characterized by the ability of the virus to elicit a strong immune response, making adenovirus-based vectors attractive vaccine carriers.121 The initial success generated from boosting a DNA vaccine combining ZEBOV GP + ZEBOV NP + SEBOV GP + ICEBOV GP with an additional booster injection of recombinant adenovirus expressing ZEBOV GP (AdHu5-ZGP) has encouraged the development of more replication-deficient adenovirus-based strategies.102 It should be noted that subsequent studies suggest that the DNA portion of this prime-boost approach is not necessary to provide protection and, indeed, is not protective when given singly.
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A single dose of AdHu5-GP mixed with AdHu5 expressing NP (AdHu5-NP) offered complete protection in NHPs against lethal ZEBOV challenge.102,122 Recently, an improved AdHu5-GP vaccine, where GP was codon optimized and under the expression of a constitutively strong chimeric promoter, was shown to fully protect infected mice when administered 30 minutes post-exposure.123 There are problems associated with the use of AdHu5-based vaccine platforms. Exposure to naturally occurring adenoviruses within the human population can lead to the development of neutralizing antibodies, potentially compromising the efficacy of adenovirus-based vaccine administration.124 Approximately 30 to 50% of the North American population and greater than 90% of the population in developing countries have pre-existing neutralizing antibodies to AdHu5. Although high doses of Adenovirus-based vaccines can circumvent pre-existing immunity, the systemic administration of high numbers of adenovirus particles (1 x 1013) can be toxic to NHPs and humans.125 Several alternatives are currently being investigated to circumvent pre-existing immunity. Ongoing research evaluating the development of adenovirus serotypes that are not as prevalent in the human population include AdHu12, AdHu35,126 and AdHu6 which was shown to be immunogenic in NHPs.127 Current efforts are also ongoing for the development of adenovirus-based vaccines originating from different animal species such as simian,124 bovine128 or porcine adenoviruses. Pre-existing immunity was by-passed in animals pre-treated with neutralizing antibodies to AdHu5 and then immunized with a vaccine based on chimpanzee adenovirus (AdC7) expressing ZEBOV GP.124 Alternatively, a bovine adenovirus subtype 3 (BAd3)based vaccine protected AdHu5 pre-exposed mice from a lethal challenge of avian influenza.128 Other studies demonstrated successful evasion of pre-existing immunity in mice by altering the routes of administration from intramuscular or oral to an intranasal mucosal vaccine delivery route.129,130 Vesiculovirus (VSV)-based candidate vaccines. Candidate vaccines based on replication competent recombinant VSV can grow to high titers and induce a strong humoral and cellular response in humans.131 VSV expressing ZEBOV antigens have been made by manipulation of an infectious VSV cDNA clone. Mice vaccinated and boosted with recombinant VSV expressing ZEBOV GP (VSV-GP) survived a challenge of ZEBOV with complete protection.132 Full protection of ZEBOV challenged mice vaccinated with VSV-GP was achieved when administered through the I.M., I.P., or mucosal route.133 When NHPs were challenged with ZEBOV following vaccination with VSV-GP, 100% protection was observed.133 Importantly, VSVbased vaccine platforms have also been shown to be an effective post-exposure treatment regimen. Mice and guinea pigs challenged with ZEBOV were administered VSV-GP 24 hours postinfection resulting in 100% and 50% protection, respectively.134 Fifty percent protection was achieved in NHPs immunized with the recombinant VSV-GP, 20–30 minutes post-exposure to ZEBOV.134 Progress has been made using the VSV-based platform for single dose blended vaccines, capable of protection against
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several EBOV species. Recombinant VSV expressing SEBOV GP, ZEBOV GP or ICEBOV GP were administered in equal concentrations as a single dose to NHPs. Full survival and protection was observed in the animals administered the blended vaccine following challenge with SEBOV, ZEBOV or ICEBOV.135 Recombinant VSV-GP vaccine given 28 days before challenge either I.N., orally (OR) or I.M. protected NHPs against a lethal challenge of ZEBOV.136 ZEBOV GP-specific T- and B-cell responses were induced in the I.N. and OR groups. These groups also produced the most IFNγ and IL-2 secreting cells, in addition to long term memory responses following immunization and challenge.136 Live attenuated recombinant vaccine vectors can provide an advantage since they elicit strong humoral and cellular immune responses.43 The protective efficacy and absence of obvious side effects of the VSV vaccine vector have been demonstrated in the mouse,132 guinea pig and NHPs models.133 Since VSV-based vaccines are replication competent recombinant viruses, questions have been raised regarding their suitability for use in humans. To address these concerns, the safety profile of VSV-GP was evaluated in NHPs infected with simian-human immunodeficiency virus (SHIV). The animals showed no evidence of vaccine-associated illnesses, and 4 of 6 SHIV-infected NHPs were protected from ZEBOV challenge, suggesting that although perhaps less efficacious, this vaccine may be safe for use in immunocompromised individuals.137 Recombinant human parainfluenza virus 3 (HPIV-3)-based candidate vaccines. Human parainfluenza virus 3 (HPIV-3) vectored systems have been shown to safely induce protection, where I.N. administration of this replication-competent respiroviruses induced a protective local and systemic immune response in guinea pigs.131,138 Preliminary experiments indicated that these vaccines could also protect NHPs. The replication-competent nature of these vectors, coupled with prevalence of antibodies against HPIV-3 due to its considerable immunity amongst the adult human population, is seen as two potential disadvantages to this vaccine candidate.138 I.N. dose of recombinant HPIV-3 expressing ZEBOV GP (HPIV-3-GP) or ZEBOV GP + NP (HPIV-3-GP + NP) were both sufficient to protect 100% of guinea pigs following a ZEBOV challenge.138 Interestingly, despite the fact that replication of the vaccine in the respiratory tract of HPIV-3-immune guinea pigs was not detected; possibly due to neutralizing antibodies in addition to cellular or other components of the immunity against HPIV-3, high titres of EBOV specific antibodies at a level slightly less than that of HPIV-3-naive animals was noted.139 NHPs vaccinated with HPIV-3-GP or HPIV-3-GP + NP followed by challenge with ZEBOV resulted in 100% protection via I.N. vaccination and 50% protection via intratracheal (I.T.) immunization.140 Routes of Vaccination and Ease of Administration Administration of a vaccine by different routes can have a significant effect on the type and strength of the induced immune response. Parenterally administered vaccines typically stimulate systemic responses, while mucosal vaccination is capable
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of inducing both systemic and mucosal immune responses.146,147 Mucosal vaccination has also been shown to overcome barriers of parenteral immunization, which is generally caused by preexisting systemic immunity to the vaccine carrier from prior exposure through natural infections or prior vaccinations.148,149 Currently, I.M. injection is the primary method of administration of filovirus vaccine candidates in addition to newly approved vaccines against other pathogens.102,133,150,151 While I.M. injection ensures that the entire vaccination dose is delivered and has proven successful in promoting strong systemic responses, the use of needles poses significant safety risks for healthcare providers and patients. Extensive training programs are usually necessary for vaccination campaigns to reduce this risk, but may be more difficult to implement in an outbreak situation due to a lack of preparation time. Alternatively, needle-free deliveries including mucosal immunization are attractive vaccination strategies. Vaccines delivered by needle-free routes will circumvent the risks associated with needle delivery, alleviate the cost and time associated with training, and potentially lead to self-vaccination. Needlefree delivery generally include the subcutaneous and transdermal routes, as well as through mucosal surfaces, which include primarily I.N. or OR administration. These routes of delivery are supported by the World Health Organization, the Global Alliance for Vaccine and Immunization, as well as the Center for Disease Control and Prevention.152 These alternative delivery methods often generate lower immune responses than I.M. vaccination and consequently most of them are still in development stages. To date, only mucosal immunization has been commonly and successfully used as an alternative to I.M. injection in vaccination programs.151,153,154 Currently, several vaccine candidates designed to afford protection to various pathogens have been delivered by the intranasal route as Ad- or VSV-based vaccines.155-162 In addition, HPIV-3 vector expressing EBOV GP conferred full protection in guinea pigs and NHPs following I.N. vaccination.138 Results from many studies indicate that both Ad- and VSV-based vectors are capable of crossing the respiratory tract to induce strong cellular and humoral immune responses. To date, nasal administration of Ad- or VSV-based EBOV vaccines have been shown to fully protect mice against lethal challenge with mouseadapted ZEBOV.129,163 A recent report has confirmed that VSVZEBOV GP has the ability to confer full protection of NHPs immunized by the I.N. or the OR route.136 Such discoveries encourage the design and use of mucosal vaccination strategies as an alternative to the traditional I.M. immunization, especially in the context of outbreaks or bioterrorism. In addition, these studies may provide new insight into the types of immune responses that are sufficient to promote survival against EBOV infection. Pharmacology and Stability Currently, the pharmacology and toxicity of EBOV vaccines is still not well understood and will need to be evaluated as more vaccine candidates get closer to clinical trials. The primary
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component of current vaccine candidates contains either the pathogen of interest in an attenuated or inactivated form, a purified protein subunit of the pathogen, or a delivery mechanism capable of producing the antigenic components of the target pathogen.164-167 Vaccines may also contain other components in their formulation, including adjuvants to induce a desired immune response and inactive components called excipients to aid in stabilization of the active component of the vaccine, which helps in its storage or delivery. Since formulation and storage may directly or indirectly alter the pharmacology and toxicity of the vaccine, the choice of formulation components is dependent on the route of administration. Therefore, choosing the correct formulation is essential for successful delivery of the active vaccine to the desired location. Other formulation components may also be necessary to improve the stability and delivery of vaccines. Since I.M. injection has a proven track record of stimulating a strong systemic immune response, it is important to ensure proper vaccine storage and stability prior to delivery. Stabilizers such as gelatin, sorbitol, sugars or alcohol sugars may be good candidates for storage and delivery of I.M. vaccines,168 but would need to be formulated depending on the type of vaccine. Mucosally delivered vaccines must be formulated to maximize stability and promote delivery to the targeted epithelium. Vaccines targeted to the nasal epithelium should be formulated with components that aid in residence time by binding mucus or the epithelium itself, and components that compromise the integrity of the airway epithelia, thereby encouraging access to the nasal associated lymphoid tissue. Non-lipid cations, cholesterol-based cationic lipids or non-cholesterol based cationic lipids have been shown to be good candidates for binding of the vaccine to the negatively charged airway epithelia, and to increase residence time in the nasal mucosa.169 A formulation including surfactants can help in delivering the vaccine to its target tissue since it temporarily compromises the integrity of the airway epithelia. Surfactants are classed by their ionic charge and are water or oil soluble. Examples that have had success in nasal delivery and are currently commercially available include polysorbate 80 and sorbitan triolate, which are components from the vaccine adjuvant MF59,170 and the surfactants Exosurf and dipalmitoylphosphatidylcholine (DPPC).171 The biggest obstacle to oral vaccination is to formulate a vaccine capable of remaining stable through the gut before being delivered to the intestinal epithelium. Therefore, an appropriate excipient must first be chosen to unite the vaccine and its components into a form suitable for oral delivery in order to increase the chances of the entire dose being delivered to the intestinal epithelium. Such excipients are called binders and include substances such as gelatin, sucrose, cellulose, polyvinylpyrrolide, starch or polyethelene glycol (PEG).168,172 Polymers such as polylactide-co-glycolide acid (PLGA) or chitosan may become more relevant for oral delivery, as several have been shown to efficiently incorporate potential antigens into the polymer matrix and maintain integrity through the gut, although delivery of their contents to the intestinal epithelium has not been shown consistently.173 Chitosan has also been shown to prolong gastric transit time, allowing greater opportunity for absorption of the vaccine.174
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Adjuvant selection also needs to be considered when formulating vaccines. Commonly used adjuvants include oil emulsions, virosomes, immunostimulation complexes (ISCOMs), microbial derivatives or aluminum based substances.175 Ad-based vaccines have been shown to produce strong T- and B-cell responses to the transgene, and therefore already contain a powerful adjuvant within the vaccine vector.121 Currently, the immunological correlates of protection necessary for survival upon filovirus infection may need to be better understood before selecting an adjuvant for the vaccine candidates. Correlates of Immune Protection In order for vaccines to be licensed they have to be assessed for toxicity, immune responses and efficacy in pre-clinical animal studies, clinical safety studies (Phase I and II), as well as clinical efficacy studies (Phase III). The goal of clinical efficacy studies is to compare vaccinated groups against placebo groups, and then compare incidence rates among the two populations. Unfortunately, such testing for EBOV vaccine candidates is difficult because there is no specific population easily identifiable that constitutes a prime target for a natural outbreak. While a study detailing the efficacy of an EBOV vaccine compared to the placebo during an outbreak situation may provide data with statistical power, some regulatory agencies have created an alternative licensing pathway for vaccine and pharmaceuticals that target highly lethal pathogens (e.g., the “animal rule”). The animal rule allows for drug approval based on data from animal efficacy, human safety and human immunogenicity research that support presumptive correlates of protection initially defined by the animal model studies. The animal rule is only used as a pathway for regulatory review when there is no other moral or feasible pathway to license a vaccine.176 The key to licensing through the animal rule is to determine correlates of immune protection that are indicative of survival in the tested animal models, and to confirm that these immune responses are indeed preserved in the immunological data obtained from humans immunized with the vaccine candidate.177 This is currently an important priority for the field to address in order to license an EBOV vaccine: to identify parameters of primary immunity in animals that predict immunological protection against infection, and to test these parameters in human immunological studies.177 To date, mouse and guinea pig models have resulted in data supporting EBOV virus-specific ELISA IgG as a quantifiable correlate of vaccine protection through DNA- and VEE-based vaccines.101,120,178 NHPs immunized with Ad-ZEBOV GP exhibited antigen specific ELISA IgG in survivors.102 Results obtained from vaccinated NHPs with VSV-based vector, parainfluenza virus vector, or VLP-based vector also induced the presence of antigen specific antibodies in all survivors prior to lethal EBOV virus challenge.118,133,140 While evidence presented to date indicates that each vector platform capable of inducing significant antibody responses is correlated to their efficacy, some subjects found to contain similar ELISA-based IgG levels have resulted in different survival outcomes. In addition, passive transfer of immune
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sera to NHPs has not been protective indicating that while ELISA-based antibody levels may be somehow indicative of protection, they are more a consequence of the protective immune response rather than its cause. The role for CD4 + and CD8 + T cells has also been documented,116,179-181 and NK cells appear to complement vaccineinduced EBOV antibodies in vaccine studies by facilitating viral clearance in mice.117 However, current methods for precise quantification of these specific cellular responses are lacking and more sensitive methods may be required before their respective role in protection are fully defined. Several groups are currently contributing to the characterization of the cellular response, by investigating different T-cell subsets in the effector and memory compartment of vaccinated animals and analyzing these responses in correlation with survival following a lethal challenge with EBOV. Better characterization of the cellular response, coupled with the advancing knowledge on the humoral response should provide tools to predict protection from immune parameters with reasonable accuracy. Conclusions Conventional heat, formalin or γ-irradiation inactivated EBOV vaccines were the first types of vaccines developed. While relatively easy to manufacture and produce, these vaccines have fallen short as they were not successful in the NHPs model. There is a stigma attached to using attenuated EBOV vaccines for fear of retention of virulence or possible reversion to an infectious state, however with genetic engineering, effective vaccines have been demonstrated to be safe and efficient in the rodent models. DNAbased vaccines provide an easily manufactured, cost effective and temperature stable vaccine alternative. Technologies for DNA delivery by gene gun or electroporation of muscle tissue can be effective, albeit physically uncomfortable, and have elicited good References 1. Feldmann H, Geisbert TW, Jahrling PB. Virus Taxonomy: VIIIth Report of the International Commitee on Taxonomy of Viruses In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, eds. Filoviridae. London: Elsevier/Academic Press 2004. 2. Bowen ET, Platt GS, Lloyd G, Raymond RT, Simpson DI. A comparative study of strains of Ebola virus isolated from Southern Sudan and northern Zaire in 1976. J Med Virol 1980; 6:129-38. 3. Buchmeier MJ, DeFries RU, McCormick JB, Kiley MP. Comparative analysis of the structural polypeptides of Ebola viruses from Sudan and Zaire. J Infect Dis 1983; 147:276-81. 4. Cox NJ, McCormick JB, Johnson KM, Kiley MP. Evidence for two subtypes of Ebola virus based on oligonucleotide mapping of RNA. J Infect Dis 1983; 147:272-5. 5. Kiley MP, Bowen ET, Eddy GA, Isaacson M, Johnson KM, McCormick JB, et al. Filoviridae: a taxonomic home for Marburg and Ebola viruses? Intervirology 1982; 18:24-32. 6. Kiley MP, Wilusz J, McCormick JB, Keene JD. Conservation of the 3' terminal nucleotide sequences of Ebola and Marburg virus. Virology 1986; 149:251-4. 7. Martini GA, Siegert R. Marburg Virus Disease. Berlin: Springer-Verlag 1971.
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immune responses. Unfortunately these strategies have demonstrated low survival in NHPs challenged with ZEBOV. The DNA prime boost adenovirus vaccine was the first vaccine to successfully protect NHPs and inspired the development of many other virus vector-based platforms. Replication deficient adenovirus platforms have been successful in protecting NHPs, and are currently undergoing clinical evaluation for safety in a Phase I trial. Because some of the population has been exposed to certain adenoviruses, further development of alternate routes of vaccine administration, other human serotypes and non-human adenoviruses is currently rapidly expanding. The VSV-based EBOV vaccine was the first to demonstrate post-exposure protection, a characteristic that is invaluable in laboratory accident, outbreak and bioweapon exposure situations. Also promising, HPIV-3 vectored systems have been shown capable of protecting NHPs from lethal ZEBOV challenge after I.N. immunization. Recently, VLP-based Ebola vaccine was also shown to protect NHPs against ZEBOV. There are several advantages of VLPs, they are non-infectious, antigens are presumably generated in their native form, they do not carry addition genetic information that is not of EBOV origin, and there would be no pre-existing immunity to VLPs. Overall, the recent past has seen tremendous advances in the development of several promising vaccine strategies to prevent fatal outcome from EBOV infection. Each strategy may offer unique advantages for specific situations such as outbreak responses, companionate use in post-exposure interventions or protection of laboratory workers or first responders. The most advantageous route of vaccination and optimal formulation will further improve these vaccines and make them optimal for certain use in the near future. Acknowledgements
The authors would like to thank Ami Patel and Gary Wong for their advice throughout the preparation of this review.
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