rVSVΔG-ZEBOV-GP (also designated V920) recombinant vesicular stomatitis virus pseudotyped with Ebola Zaire Glycoprotein: Standardized template with key considerations for a risk/benefit assessment.

The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety and characteristics of live, recombinant viral vector vaccines. A recent publication by the V3SWG described live, attenuated, recombinant vesicular stomatitis virus (rVSV) as a chimeric virus vaccine for HIV-1 (Clarke et al., 2016). The rVSV vector system is being explored as a platform for development of multiple vaccines. This paper reviews the molecular and biological features of the rVSV vector system, followed by a template with details on the safety and characteristics of a rVSV vaccine against Zaire ebolavirus (ZEBOV). The rVSV-ZEBOV vaccine is a live, replication competent vector in which the VSV glycoprotein (G) gene is replaced with the glycoprotein (GP) gene of ZEBOV. Multiple copies of GP are expressed and assembled into the viral envelope responsible for inducing protective immunity. The vaccine (designated V920) was originally constructed by the National Microbiology Laboratory, Public Health Agency of Canada, further developed by NewLink Genetics Corp. and Merck & Co., and is now in final stages of registration by Merck. The vaccine is attenuated by deletion of the principal virulence factor of VSV (the G protein), which also removes the primary target for anti-vector immunity. The V920 vaccine caused no toxicities after intramuscular (IM) or intracranial injection of nonhuman primates and no reproductive or developmental toxicity in a rat model. In multiple studies, cynomolgus macaques immunized IM with a wide range of virus doses rapidly developed ZEBOV-specific antibodies measured in IgG ELISA and neutralization assays and were fully protected against lethal challenge with ZEBOV virus. Over 20,000 people have received the vaccine in clinical trials; the vaccine has proven to be safe and well tolerated. During the first few days after vaccination, many vaccinees experience a mild acute-phase reaction with fever, headache, myalgia, and arthralgia of short duration; this period is associated with a low-level viremia, activation of anti-viral genes, and increased levels of chemokines and cytokines. Oligoarthritis and rash appearing in the second week occur at a low incidence, and are typically mild-moderate in severity and self-limited. V920 vaccine was used in a Phase III efficacy trial during the West African Ebola epidemic in 2015, showing 100% protection against Ebola Virus Disease, and it has subsequently been deployed for emergency control of Ebola outbreaks in central Africa. The template provided here provides a comprehensive picture of the first rVSV vector to reach the final stage of development and to provide a solution to control of an alarming human disease.

Introduction

Recombinant viral vectors expressing heterologous antigens (and antibodies) represent promising platforms for developing novel vaccines and therapies against human and animal infectious diseases and cancers. Development of new viral vectors, remodeling of vector backbones to improve their biological activity, and incorporation of new foreign proteins in existing vector platform result in unique viral products requiring assessments of safety, innate and adaptive immune response, manufacturability and the regulatory pathway. It is important to understand how chimeric viral vectors differ from the wild-type progenitors, based on modifications within the vector backbone and the effect of adding a heterologous gene, which may influence pathogenesis. This is particularly true for replicating, attenuated vaccines (as distinguished from replication defective vectors (e.g. adenoviruses, alphavirus replicons, and herpes simplex viruses), or live vectors that are attenuated due to host range restriction (e.g. Modified Vaccinia Ankara, Sendai, and Newcastle disease viruses). In general, replicating vaccines have proven most effective in generating rapid and durable protection against viral infections [1]. A number of rationally developed, recombinant, replicating, attenuated viral vector vaccines are in clinical development, and a few are nearing licensure or have reached commercialization. Among the prominent platforms for constructing such vaccines are vaccinia virus, (veterinary applications) [2]; measles virus [3]; adenovirus type 4 [4]; alphaviruses, such as Sindbis and Venezuelan equine encephalitis virus [5]; flaviviruses, including dengue virus and yellow fever virus 17D [6]; cytomegalovirus [7]; and vesicular stomatitis virus [8], the subject of this paper.

The Brighton Collaboration (www.brightoncollaboration.org) was formed in 2000 as an international voluntary collaboration to enhance the science of vaccine safety research [9]. In recognition of these needs in this domain, the Brighton Collaboration created the Viral Vector Vaccines Safety Working Group (V3SWG) in October 2008. Analogous to the value embodied in standardized case definitions for Adverse Events Following Immunization (AEFI), the V3SWG believes a standardized template describing the key characteristics of a novel vaccine vector, when completed and maintained with the latest research, will facilitate the scientific discourse among key stakeholders by increasing the transparency and comparability of information. The International AIDS Vaccine Initiative (IAVI) had already developed an internal tool to assess the risk/benefit of different viral vectors under its sponsorship. The IAVI graciously shared this tool with the V3SWG for adaptation and broader use as a standardized template for collection of key information for risk/benefit assessment on any viral vector vaccines. This tool was aimed at identifying potential major hurdles or gaps that would need to be addressed during the development of vectored vaccines. The template collects information on the characteristics of the wild type virus from which the vector was derived as well as known effects of the proposed vaccine vector in animals and humans, manufacturing features, toxicology and potency, nonclinical studies, and human use, with an overall adverse effect and risk assessment.

Following the process described above and on the Brighton Collaboration Website (http://cms.brightoncollaboration.org:8080/public/what-we-do/setting-standards/case-definitions/process.html), the Brighton Collaboration V3SWG was formed in October 2008 and includes ∼15 members with clinical, academic, public health, regulatory and industry backgrounds with appropriate expertise and interest. The composition of the working and reference group, as well as results of the web-based survey completed by the reference group with subsequent discussions in the working group, can be viewed at http://www.brightoncollaboration.org/internet/en/index/workinggroups.html. The workgroup meets via emails and monthly conference calls coordinated by a secretariat [9].

The V3SWG anticipates that eventually all developers/researchers of viral vector vaccines (especially those in clinical development) will complete the template and submit it to the V3SWG and Brighton Collaboration for peer review and eventual publication in Vaccine. Following this, to promote transparency, the template will be posted and maintained on the Brighton Collaboration website for use/reference by various stakeholders. Furthermore, recognizing the rapid pace of new scientific developments in this domain (relative to AEFI case definitions), we hope to maintain these completed templates “wiki-” style with the help of Brighton Collaboration and each vectored vaccine community of experts [10].

Vesicular stomatitis virus (VSV) as a platform for recombinant, replicating vaccines

This paper is preceded by a recent V3SWG and Brighton Collaboration publication by Clarke et al. [8] describing the history and rationale for development of VSV as a replicating vector platform; the natural history of parental VSV viruses; the construction of recombinant vaccines pseudo-typed with heterologous proteins that stimulate humoral and cellular immunity; attenuation strategies; the application of animal models to test safety and efficacy; manufacturing; and the status of clinical development. The underlying principles explained by Clarke et al. [8] are critical background to the present paper. A number of general points are re-emphasized below, and additional background is provided to facilitate an understanding of the recombinant rVSV platform as applied to the development of vaccines against viral hemorrhagic fevers. These comments are followed by the template which provides specific features of the most advanced rVSV vector in development, a vaccine against Ebola virus disease in which the VSV glycoprotein (G) is entirely deleted and replaced with the corresponding glycoprotein (GP) of the Zaire Ebolavirus (rVSVΔG-ZEBOV-GP). This vaccine, currently designated V920, is being developed by Merck & Co., Kenilworth, NJ, USA and is in the registration process.

Aspects of the rVSV technology of special interest for vaccine development

VSV (a negative sense, single-strand RNA virus belonging to the family Rhabdoviridae, genus Vesiculovirus) is being widely explored for vaccine development against infectious diseases and cancer, and as an oncolytic virus.

VSV causes self-limited disease in horses, pigs, and cattle, and may be either asymptomatic or cause a mild flu-like syndrome in humans and is thus a naturally attenuated vector backbone for development of human vaccines and therapies.

Other advantages of VSV as a vector include (i) low prevalence of immunity to the vector in most populations targeted for immunization; (ii) the viral RNA does not integrate into the host, posing little risk of oncogenesis or mutagenesis; (iii) large foreign transgenes can be packaged and expressed; (iv) the virus may be pseudo-typed with heterologous viral glycoproteins presented in the envelope in their natural conformation.

There are two major VSV serotypes (VSV-Indiana and VSV-New Jersey). VSV-Indiana (VSV-I) is the basis for current vaccine candidates. Other related vesiculoviruses, such as Isfahan virus [11] and Maraba virus [12], and more distantly related rhabdoviruses, such as rabies virus are also being explored as viral vectors [13].

The VSV genome consists of ∼11,000 nucleotides encoding five major proteins. The VSV glycoprotein (G) located in the viral envelope is responsible for attachment to cells, fusion with endosomal membranes at low pH and release of viral genomic RNA into the cytoplasm. The G protein also elicits protective immunity against VSV.

Using reverse genetic systems, VSV vectors have been constructed expressing genes from divergent species, including many viruses (e.g. Ebola virus, Marburg virus, Lassa fever virus, HIV, influenza virus, EV71, HPV and others, see template), bacteria [14], and tumor antigens [15]. In some constructs, a portion of the VSV G protein is retained to facilitate expression or enable fusion and internalization of the recombinant virus [8]. VSV vectors completely lacking the VSV G gene (VSVΔG) must reconstitute the attachment, fusion and budding (release) functions with one or more proteins encoded by the heterologous envelope gene. In the case of rVSVΔG-influenza, for example, VSV G was replaced with influenza hemagglutinin (HA), neuraminidase (NA) or both; only virus expressing both HA and NA in the same vector produced replication-competent pseudo-type virus [16], since both proteins play a role in attachment and because NA is needed for virus release from host cells. Similarly, in the case of a henipavirus (Nipah), a pseudo-type expressing the Nipah glycoprotein (G) responsible for cell attachment did not produce replicating virus unless a fusion protein [F protein of Nipah or the glycoprotein (GP) of Ebola Zaire] was coexpressed [17].

Replicating rVSVΔG pseudotypes with glycoprotein (GP) derived from many different filoviruses [Ebola zaire, Ebola sudan, Ebola reston, Marburg, Bundibugyo, Tai Forest, and Lloviu have been constructed [18], [19], [20], with the GP providing virus attachment and class I fusion functions. The most advanced vaccine candidate described in this template is rVSVΔG-ZEBOV-GP expressing Zaire Ebola virus (ZEBOV) GP in place of the VSV-I G protein.

The reverse genetics system producing rVSVΔG-ZEBOV-GP involves co-transfection of cells with plasmids containing the entire VSV genome with G deleted and replaced with ZEBOV GP, together with helper plasmids expressing the VSV N, P, and L genes [28]. Transcription of the plasmids is controlled by bacteriophage T7 polymerase supplied by baby hamster kidney cells expressing T7 (as done for rVSVΔG-ZEBOV-GP) or exogenously by a recombinant vaccinia expressing T7 polymerase.

The rVSVΔG-ZEBOV-GP is constructed with full-length GP anchored in the viral envelope, whereas native ZEBOV expresses an abundant soluble form of GP without the transmembrane domain (soluble GP, sGP), which may act as a decoy for antibody contributing to evasion of neutralizing antibody during filovirus infection [21]. As, rVSVΔG-ZEBOV-GP generates no sGP it is more efficiently neutralized by antibody than wild-type ZEBOV [22].

The full length heterologous GP is incorporated into the rVSV particle, which retains typical bullet shaped morphology, the viral envelope being decorated with ZEBOV GP spikes instead of VSV G protein spikes. The GP spike is composed of disulfide linked subunits, GP1 and GP2. Three GP1 subunits form a 3-bladed propeller-like trimer consisting of the receptor binding domains, glycosylated mucin-like domains and glycan caps. The glycans are hypothesized to shield epitopes from neutralizing antibody [23], [24]. However this is uncertain, since neutralization can occur prior to cleavage of the mucin-like domain in the endosome. Moreover, a mutated rVSVΔG-ZEBOV-GP lacking GP1 glycans was not more efficient in eliciting neutralizing antibodies in mice [26].

In standard EM studies, insertion of Ebola GP into rVSV particles did not alter typical bullet-shaped vesiculovirus morphology. However, while the structure of the ZEBOV GP has been partially resolved by cryo-EM at high resolution [23] that of GP in pseudo-typed VSV has not been elucidated.

The cell targets for infection, determined by virus ligand-cell receptor interactions, may differ for virus pseudo-typed with ZEBOV GP compared to natural VSV, although there may be overlapping tropisms. Certain cell lines susceptible to VSV but not ZEBOV, such as Jurkat cells and insect cells, do not permit rVSVΔG-ZEBOV-GP replication [18], [26]. The primary in vivo ZEBOV targeted cells are thought to be endothelial cells, monocytes, macrophages, and myeloid dendritic cells [27]. Although this is also presumed to hold true for the pseudo-typed rVSV, there is no systematic study of the cell types productively infected by rVSVΔG-ZEBOV-GP in vivo. Consistent with ZEBOV GP-specific tropism, a limited number of observations suggest that endothelial cells are a target for rVSVΔG-ZEBOV-GP [28] and a biodistribution study in macaques showed that the vaccine virus targeted lymphoreticular tissues [Merck & Co., Inc., Kenilworth, NJ, USA, NewLink Genetics Corp, unpublished data, 2017]. Interestingly, a study in swine (unpublished, described in the template) showed that rVSVΔG-ZEBOV-GP induced self-limited clinical disease, histopathology and cell tropism, similar to that induced by wild-type VSV. Although pigs are also susceptible to ZEBOV by the respiratory route [29], pathogenesis is distinct from that caused by VSV. Thus, in swine, the pathogenesis of the host-virus pairing appears to match that of the vector backbone rather than the donor of the heterologous envelope. Possibly, the retention of the intact VSV M gene, a virulence factor of VSV [30], in the recombinant rVSVΔG-ZEBOV-GP vector plays a role in pathogenesis for swine. These observations provide fertile ground for future research in mechanisms of viral pathogenesis.

Both wild type ZEBOV and rVSV pseudo-typed with ZEBOV GP appear to enter the cell by macropinocytosis in a GP protein-dependent manner [31]. The cell receptors initiating this process remain poorly defined and entry does not appear to involve clathrin (in contrast, VSV G protein binds low-density lipoprotein receptors and enters via receptor-mediated endocytosis by a clathrin-dependent pathway [32]). C-type lectins (e.g. DC-SIGN) are putative cell surface receptors for ZEBOV GP [33]. However, a critical virus-cell receptor interaction is intracellular, an important consideration for immune recognition. Once in the endosome, proteolytic processing is initiated by cathepsin proteases, after which GP1 binds to receptors on endosomal membranes, predominantly the Niemann-Pick C1 protein [34]. Cleavage of the mucin-like domain and the glycan cap on G1 are required for receptor binding to occur [23]. At low pH, conformational rearrangement of GP2 exposes a hydrophobic fusion loop which is inserted into the endosome membrane followed by internalization of the viral RNA. Neutralization of Ebola virus by antibody involves multiple different mechanisms, including blocking cathepsin-mediated proteolytic cleavage of GP1, blocking binding of GP1 to Niemann Pick C1 receptors, and inhibition of GP2 mediated fusion [35], [36].

Since the VSV G gene is deleted in rVSVΔG-ZEBOV-GP, anti-vector immunity is minimized as a factor for primary immunization or sequential use of vectors expressing different heterologous genes.

Complete deletion of VSV G protein and replacement by the heterologous transgene, as in rVSVΔG-ZEBOV-GP, results in a highly attenuated phenotype. Removal of VSV G, the principal virulence factor, is critical to this attenuation, since constructs retaining G or a portion thereof show varying degrees of residual neurovirulence when injected directly into the brain of young mice [8].

Attenuation of the rVSVΔG-ZEBOV-GP vaccine candidate has been extensively studied. Neurovirulence is a feature of parental VSV infection following intracranial injection of most animal species (see template, Table 2), whereas the rVSVΔG-ZEBOV-GP virus is pathogenic only for infant (not adult) mice and caused minimal histopathology without clinical signs after intracranial inoculation in non-human primates [37]. No clinical, biochemical or pathoanatomical effects were observed in mice, rats, and nonhuman primates in formal toxicology studies where the vaccine was administered IM at the full human dose (see template). The rVSVΔG-ZEBOV-GP vaccine (V920) has now been administered to over 20,000 persons in Phase 1–3 and expanded access clinical trials and has been shown to have a favorable safety profile and to be generally well tolerated.

Table 2

Standardized template.

Risk/benefit assessment for vaccine vectors
1. Basic Information	Information
1.1. Author(s)	Thomas P. Monath M.D., Patricia E. Fast M.D., Ph.D. Kayvon Modjarrad, M.D., Ph.D., David Clarke, Ph.D., Brian K. Martin Ph.D., Joan Fusco Ph.D., R. Nichols M.S., D. Gray Heppner M.D., Jakub K Simon, M.D., M.S., Sheri Dubey, M.S., Sean P. Troth D.V.M, Ph.D., Jayanthi Wolf, Ph.D., Beth-Ann Coller, Ph.D.
1.2. Date completed/updated	April 2018
2. Vaccine Vector information	Information
2.1. Name of Vaccine Vector	rVSVΔG-ZEBOV-GP (also designated V920). Recombinant Vesicular Stomatitis Virus Pseudo-typed with Ebola Zaire Glycoprotein
2.2. Class/subtype	Live, attenuated replication competent viral vector
2.3. Proposed route of administration	Intramuscular (IM)
3. Characteristics of wild type agent	Information	Comments/Concerns	Reference(s)
3.1. Please list any disease(s) caused by wild type, the strength of evidence, severity, and duration of disease for the following categories:

VSV pseudo-types can propagate to high titers in mammalian cells, although some degree of attenuation is observed compared to wild-type VSV [18]. As VSV is an interferon-sensitive virus, interferon deficient cells, such as Vero cells, are particularly productive. Vero cells are widely used for manufacturing other vaccines, including multiple licensed, live attenuated vaccines (e.g. poliovirus, smallpox virus, rotavirus, dengue virus). The rVSVΔG-ZEBOV-GP virus grows to ∼8.0 log10 plaque-forming units/mL (PFU/mL) in Vero cells without serum or animal derived components. The virus can then be clarified by filtration and purified and concentrated by a straightforward process involving enzyme digestion and ultrafiltration/diafiltration without chromatography and with minimal product loss. This manufacturing process has been up-scaled to produce large quantities of vaccine and is undergoing validation at a dedicated facility.

Many live, replicating vaccines are susceptible to thermal instability and require lyophilization for long term storage. Because of the short development time for rVSVΔG-ZEBOV-GP during the West African Ebola emergency in 2014–2015, the vaccine was produced and stored in unit dose containers as a frozen liquid formulation, stored at ≤−60 °C. Interestingly, the vaccine was found to be stable when thawed and held at 2–8 °C for at least 2 weeks, a feature that facilitates distribution for use in control of outbreaks.

The rVSVΔG-ZEBOV-GP vaccine has been extensively tested in nonhuman primates with respect to immunogenicity and protective efficacy (see template, Table 2). The published literature has been supplemented by multiple additional studies, largely aimed at determining the immune correlate(s) of protection which may then be bridged to human immune responses elicited by the rVSVΔG-ZEBOV-GP vaccine. These efforts are ongoing. In one study, IgM subclass antibodies were suggested to play a dominant role in rVSVΔG-ZEBOV immunity compared to IgG [38] but this observation has not been further investigated.

Inoculation of rVSVΔG-ZEBOV-GP is followed by rapid appearance of viremia and activation of innate immune responses, including NK cells, which are believed to be at least partially responsible for protection against challenge given 3–7 days after vaccination [43], [44], [45], [46], [47], [48] or shortly before vaccination (i.e. post exposure vaccination [113]). GP-specific IgG antibodies appear between 7 and 14 days after vaccination and tend to peak at 28 days. A signature of innate immune markers appearing during the first few days after vaccination was found to correlate with antibody levels appearing later. Predominant among the independent markers were IP-10 and CXCR6 expression on NK cells on day 1 as independent correlates. These observations are consistent with other live viral vaccines, such as yellow fever vaccine, showing predictive innate signatures that shape the adaptive response [39].

The role of neutralizing antibodies in protection elicited by rVSVΔG-ZEBOV-GP vaccine remains uncertain. The vaccine elicits robust neutralizing antibody responses following vaccination as measured by plaque reduction with the homologous (vaccine) virus, or in a ZEBOV pseudo-virion assay but neutralization titers against wild-type virus appear to be low [47]. The repertoire of antibodies elicited by the vaccine is not yet known, but it is clear that neutralizing monoclonal antibodies are highly protective; passive immunization with certain monoclonal antibodies can abrogate infection and prevent illness/death in the NHP model even when given up to 5 days after challenge [40]. In addition to neutralization, non-neutralizing antibodies with functional activities, including ADCC [40] and phagocytosis are probable secondary mediators; moreover, cooperative effects of non-neutralizing antibodies may enhance the potency of neutralizing antibodies [41].

There have been few studies of T cell responses in NHPs or humans vaccinated with rVSVΔG-ZEBOV-GP. However, the vaccine does not appear to elicit robust T cell responses in NHPs [42]. Moreover, T cell depletion studies in vaccinated NHPs indicated that CD8+ T cells did not play a role in protection [42]. In humans, broad T cell activation was observed by Day 7 after vaccination, but ZEBOV specific cytotoxic CD8+ T cell responses were seen only at the higher vaccine dose (2 × 107 pfu) [43], consistent with antibodies being the predominant mediator of protection.

A substantial experience has now accumulated on the safety, immunogenicity and protective efficacy of rVSVΔG-ZEBOV-GP in humans (Table 1). It is remarkable that this effort was carried out by a coalition of multiple international partners over a short period of time and during an international public health emergency in West Africa [44]. The logistical problems and solutions encountered in one of the large trials are chronicled in a recent series of publications [J Infect Dis 2018:217 (Suppl. 1)].

Table 1

Table of All Clinical Studies of rVSVΔG-ZEBOV-GP vaccine (V920).

Study ID	Phase	Country	Study title	Study design*	Dosing regimen (V920, IM)*	Study population	Status of trial subject exposure+
Protocol V920-001-06 (NLG 0307; WRAIR 2163)	1	USA	A Phase 1 Randomized, Single-Center, Double-Blind, Placebo Controlled, Dose-Escalation Study to Evaluate the Safety and Immunogenicity of the BPSC1001 (V920) Ebola Virus Vaccine Candidate in Healthy Adult Subjects	Randomized, double-blind, placebo-controlled, dose-escalation	3 × 106 pfu/mL (n = 10), 2 × 107 pfu/mL (n = 10), 1 × 108 pfu/mL (n = 10), placebo (n = 9)	Healthy eligible subjects between the ages of 18 and 50 years	Completed
Protocol V920-002-04 (NLG 0207; NIH 15-I-0001)	1	USA	A Phase 1 Randomized, Double-Blind, Placebo Controlled, Dose-Escalation Study to Evaluate the Safety and Immunogenicity of Prime-Boost VSV Ebola Vaccine in Healthy Adults	Randomized, double-blind, placebo controlled, dose-escalation studyEvaluates two doses of V920	3 × 106 pfu/mL (n = 10), 2 × 107 pfu/mL (n = 10), 1 × 108 pfu/mL (n = 10), placebo (n = 9) on days 0 and 28	Healthy eligible subjects between the ages of 18 and 65 years	Completed
Protocol V920-003-01 (#CI1401, Halifax, Canada)	1	Canada	A Phase 1 Randomized, Single-Center, Double-Blind, Placebo Controlled, Dose-Ranging Study to Evaluate the Safety and Immunogenicity of the BPSC-1001 (V920) Ebola Virus Vaccine Candidate in Healthy Adult Subjects	Randomized, double-blind, placebo controlled, dose-ranging study	1 × 105 pfu/mL (n = 10), 5 × 105 pfu/mL (n = 10), 3 × 106 pfu/mL (n = 10); placebo (n = 10)	Healthy eligible subjects between the ages of 18 and 65 years	Completed
Protocol V920-004-03 (NLG 0507)	1b	USA	A Phase 1 Randomized, Multi-Center, Double-Blind, Placebo-Controlled, Dose-Response Study to Evaluate the Safety and Immunogenicity of the BPSC1001 (V920) Ebola Virus Vaccine Candidate in Healthy Adult Subjects	Randomized, multi-center, double-blind, placebo controlled, dose-ranging	3 × 103 pfu/mL (n = 64), 3 × 104 pfu/mL (n = 64), 3 × 105 pfu/mL (n = 64), 3 × 106 pfu/mL (n = 84), 9 × 106 pfu/mL (n = 47), 2 × 107 pfu/mL (n = 47), 1 × 108 pfu/mL (n = 48) placebo (n = 94)	Healthy eligible subjects between the ages of 18 and 60 years	Completed
Protocol V920-005-08 (Geneva)	1	Switzerland	A Phase 1/2 dose-finding randomized, single-center, double-blind, placebo-controlled safety and immunogenicity trial of the vesicular stomatitis virus-vectored Zaire Ebola candidate vaccine BPSC1001 (V920) in healthy adults	Randomized, single-center, double-blind, placebo-controlled, dose-finding	3 × 105 pfu/mL (n = 51), 1 × 107 pfu/mL (n = 35), 5 × 107 pfu/mL (n = 16), placebo (n = 13)	Healthy eligible subjects between the ages of 18 and 65 years	Completed
Protocol V920-006-05 (Hamburg)	1	Germany	An open label, single center, dose escalation Phase 1 trial to assess the safety, tolerability and immunogenicity of a single ascending dose of the Ebola Virus vaccine V920 (BPSC1001)	Open label, single center, dose escalation	3 × 105 pfu/mL (n = 10), 3 × 106 pfu/mL (n = 10), 2 × 107 pfu/mL (n = 10)	Healthy eligible subjects between the ages of 18 and 55 years	Completed
Protocol V920-007-04 (Gabon)	1	Gabon	A Phase 1, Randomized, Open-Label, Dose-Escalation Study to Evaluate the Safety and Immunogenicity of the BPSC1001 (V920) Ebola Virus Vaccine Candidate in Healthy Adult and Children Volunteers in Lambaréné, Gabon	Open label, dose escalation study	3 × 103 pfu/mL (n = 21), 3 × 104 pfu/mL (n = 19), 3 × 105 pfu/mL (n = 20), 3 × 106 pfu/mL (n = 39), 2 × 107 pfu/mL (n = 16)2 × 107 6 to 12 years (n = 20)2 × 107 13 to 17 years (n = 20)	Healthy eligible adults between the ages of 18 and 50 (later included a cohort of children 6 to 12 and adolescents 13 to 17 years of age)	Completed
Protocol V920-008-03 (Kenya)	1	Kenya	A Phase 1, Open-Label, Dose-Escalation Study to Evaluate the Safety and Immunogenicity of the BPSC1001 (V920) Ebola Virus Vaccine Candidate in Healthy Adult Volunteers in Kilifi, Kenya	Open label, dose escalation study	3 × 106 pfu/mL (n = 20)1 × 107 pfu/mL (n = 20)	Healthy eligible adult health workers ages 18–55 years	Completed
Protocol V920-009-05	2	Liberia	Partnership for Research on Ebola Vaccines in Liberia (PREVAIL)	Randomized, double-blind, placebo-controlled study	2 × 107 pfu/mL (n = 500)placebo (n = 500)	Subjects ≥18 years	Completedǂ
Protocol V920-010-04	3	Guinea	A Randomized Trial to Evaluate Ebola Vaccine Efficacy and Safety in Guinea, West Africa	Randomized ring vaccination	2 × 107 pfu/mLn = 5837 vaccinated, including 194 children 6–17 years of age	Subjects ≥18 years who live in the defined vaccination ringExpanded to include children 6–17 years of age in Protocol Amendment 4	Completed
Protocol V920-011-05	2/3	Sierra Leone	Sierra Leone Trial to Introduce a Vaccine against Ebola (STRIVE)	Randomized, open label	2 × 107 pfu/mLn = 8673 enrolled (7998 vaccinated)	Subjects 18 years or older who are at high risk of exposure to EVD	Completed
Protocol V920-012-02	3	USA, Canada, Spain	A Phase 3, Randomized, Placebo-Controlled, Clinical Trial to Study the Safety and Immunogenicity of Three Consistency Lots and a High Dose Lot of V920 Ebola Vaccine in Healthy Adults	Randomized, double-blind, placebo-controlled	2 × 107 consistency lot A (n = 266), 2 × 107 consistency lot B (n = 265), 2 × 107 consistency lot C (n = 266), 1 × 108 pfu/mL (n = 264), placebo (n = 133)	Healthy eligible subjects between the ages of 18 and 65 years	Completed
Protocol V920-013-03	2	USA, Canada	A Multicenter Study of the Immunogenicity of Recombinant Vesicular Stomatitis Vaccine for Ebola-Zaire (V920) for Pre-Exposure Prophylaxis (PREP) In Individuals at Potential Occupational Risk for Ebola Virus Exposure	Individuals at potential occupational risk, Randomized, Open label, booster or no booster at 18 months	Planned 2 × 107 pfu/mL (n ∼ 800)	Subjects with potential occupational risk who are 18 years and older	Ongoing
Protocol V920-015-03	2	Canada, Burkina Faso, Senegal	A Phase 2 Randomized, Multi-Center Double-Blind, Placebo-Controlled Study to Evaluate the Safety and Immunogenicity of 1 or 2 doses of the V920 Ebola Virus Vaccine Candidate in HIV-Infected Adults and Adolescents	Randomized, double-blind, placebo-controlled, one or two doses of V920	Planned 2 × 107 pfu/mL (n ∼ 200), placebo (n ∼ 50)	HIV infected adults and adolescents	Ongoing
Protocol V920-016-02	2	Guinea, Liberia, Sierra Leone	Partnership for Research on Ebola Vaccination (PREVAC)	Randomized, double-blind, placebo-controlled trial of three vaccine strategies (Ad26.ZEBOV/MVA-BN-Filo vaccine-Janssen, V920 with or without boost at 56 days	Planned 2 × 107 pfu/mL (n ∼ 1650)Placebo (n ∼ 550)	Subjects (adults and children), aged at least 1 year of age	Ongoing (first subject enrolled in version 3.0 that includes dosing with V920 on 24-Jul-2017)
Protocol V920-018-02	3	Guinea	A Randomized Trial to Evaluate Ebola Vaccine Efficacy and Safety in Guinea, West Africa	Front Line Workers, open label	2 × 107 pfu/mLn = 2115 enrolled (2016 vaccinated)	All eligible frontline workers	Completed

Note: V920-014 is a placeholder for a potential pediatric clinical trial that is indefinitely on hold. V920-017 is an expanded access trial to be used in additional Ebola outbreaks in Africa.

All studies administered a single dose of V920, except for the V920-002 trial in which 2 doses were administered; dose levels for V920 are nominal.

Status of Trial subject exposure is current as of 01-Aug-2017.

Long-term follow-up is continuing for a subset of study participants.

Key aspects of the Phase 1–3 clinical trials, which have engaged >18,000 participants are detailed in the template. Overall, when administered at the selected nominal dose of 2 × 107 pfu, rVSVΔG-ZEBOV-GP vaccine has proven to be safe and well tolerated. During the first few days after vaccination, many vaccinees experience an acute-phase reaction with fever, headache, myalgia, and arthralgia of short duration; this period is associated with a low-level viremia, activation of anti-viral genes, and increased levels of chemokines and cytokines [28], [39], [125]. Oligoarthritis and rash appearing in the second week, occur in a minority of subjects, and are typically mild-moderate in severity and self-limited. Vesicular mucosal lesions are infrequent. The arthritis and skin events appear to reflect direct viral injury and inflammation and do not have an immunopathological basis [28], [48]. As with any new vaccine, very rare adverse events may not be detected until accumulation of a large safety data base (1–3 million persons immunized).

The clinical trials have shown the vaccine to be highly immunogenic across a broad dose range of 3 × 103–1 × 108 pfu, with >95% of subjects developing IgG binding antibodies (ELISA using recombinant GP antigen) and neutralizing antibodies (using several different methods, but predominantly plaque reduction method with pseudo-typed virus, e.g. rVSVΔG-ZEBOV-GP) (Table 1, Table 2) [28], [46], [47], [48]. Lower levels of neutralizing antibody to wild-type Ebola have been observed [28] possibly due to competition with sGP. IgG and neutralizing antibodies appear between days 7–14, peak on day 28, and plateau thereafter for at least 24 months.

In healthy people

In immunocompromised

In neonates, infants, children

During pregnancy and in the unborn

Are there any other susceptible human populations

Animals

Characteristics of proposed vaccine vector

Is there a large-scale manufacturing feasibility?

IP Protection for rVSVΔG-ZEBOV-GP?

Rodent

Human

HIV

Other diseases

Healthy people?

Immunocompromised?

Neonates, infants, children?

Elderly

Pregnancy and in the unborn?

Gene therapy experiments?

Any other susceptible populations?

Healthy people?

Immunocompromised?

Neonates, infants, children?

Elderly

Pregnancy and in the unborn?

Other susceptible populations?

Local reactions (mild to moderate in intensity)

Systemic reactions (mild to moderate in intensity)

Severe local reactions

Severe systemic reactions

All studies in humans (and most in NHP) have used the IM route of administration, and there has been no comparison to subcutaneous (SC) delivery. This is simply a reflection of the rapid pace of development of the vaccine.

Efficacy of rVSVΔG-ZEBOV-GP vaccine was demonstrated in a large ring vaccination trial in Guinea in which contacts of an Ebola case and contacts of contacts were randomized to receive a single injection of 2 × 107 pfu rVSVΔG-ZEBOV-GP vaccine immediately or after a 21-day delay. [49], [50] Analysis of efficacy in the randomized rings compared all vaccinated subjects in the immediate arm (2119 subjects in 51 rings) to all eligible subjects who consented on Day 0 in the delayed arm (1435 subjects in 46 rings). Ten cases of confirmed EVD (in 4 rings) were observed in eligible subjects in the delayed vaccination arm who consented on Day 0 while no cases of EVD occurred in the vaccinated subjects in the immediate arm >10 days after vaccination. The calculated vaccine efficacy in this analysis was 100% (95% CI: 63.5–100%, p = 0.0471). This remarkable trial was conducted at the tail end of the West African epidemic and underpins the regulatory review towards licensure of the vaccine, as well as pre-approval use in controlling outbreaks of Ebola virus disease.

A summary of all clinical trials employing rVSVΔG-ZEBOV-GP is provided in Table 1.

Disclaimer

The findings, opinions, conclusions, and assertions contained in this consensus document are those of the individual members of the Working Group. They do not necessarily represent the official positions of any participant’s organization (e.g., government, university, or corporations) and should not be construed to represent any Agency determination or policy.