The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety and characteristics of live, recombinant viral vector vaccines. The Modified Vaccinia Ankara (MVA) vector system is being explored as a platform for development of multiple vaccines. This paper reviews the molecular and biological features specifically of the MVA-BN vector system, followed by a template with details on the safety and characteristics of an MVA-BN based vaccine against Zaire ebolavirus and other filovirus strains. The MVA-BN-Filo vaccine is based on a live, highly attenuated poxviral vector incapable of replicating in human cells and encodes glycoproteins of Ebola virus Zaire, Sudan virus and Marburg virus and the nucleoprotein of the Thai Forest virus. This vaccine has been approved in the European Union in July 2020 as part of a heterologous Ebola vaccination regimen. The MVA-BN vector is attenuated following over 500 serial passages in eggs, showing restricted host tropism and incompetence to replicate in human cells. MVA has six major deletions and other mutations of genes outside these deletions, which all contribute to the replication deficiency in human and other mammalian cells. Attenuation of MVA-BN was demonstrated by safe administration in immunocompromised mice and non-human primates. In multiple clinical trials with the MVA-BN backbone, more than 7800 participants have been vaccinated, demonstrating a safety profile consistent with other licensed, modern vaccines. MVA-BN has been approved as smallpox vaccine in Europe and Canada in 2013, and as smallpox and monkeypox vaccine in the US in 2019. No signal for inflammatory cardiac disorders was identified throughout the MVA-BN development program. This is in sharp contrast to the older, replicating vaccinia smallpox vaccines, which have a known risk for myocarditis and/or pericarditis in up to 1 in 200 vaccinees. MVA-BN-Filo as part of a heterologous Ebola vaccination regimen (Ad26.ZEBOV/MVA-BN-Filo) has undergone clinical testing including Phase III in West Africa and is currently in use in large scale vaccination studies in Central African countries. This paper provides a comprehensive picture of the MVA-BN vector, which has reached regulatory approvals, both as MVA-BN backbone for smallpox/monkeypox, as well as for the MVA-BN-Filo construct as part of an Ebola vaccination regimen, and therefore aims to provide solutions to prevent disease from high-consequence human pathogens. Ó 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The Brighton Collaboration (www.brightoncollaboration.org) was launched in 2000 to improve the science of vaccine safety [1] . The Brighton Collaboration formed the Viral Vector Vaccines Safety Working Group (V3SWG) in October 2008 to improve our ability to anticipate potential safety issues and meaningfully assess or interpret safety data, thereby facilitating greater public acceptance when a viral vector vaccine is licensed [2] . The V3SWG has developed a standardized template describing the key characteristics of a novel viral vaccine vector to facilitate the scientific discourse among key stakeholders and increase the transparency and comparability of information. This introduction and the ''specific instructions" provide definitions and additional guidance for completing the template (V2.0) that follows.
Viral vector vaccines are laboratory-generated, chimeric viruses that are based upon replicating or non-replicating virus vectors into which have been spliced genes encoding antigenic proteins for a target pathogen. Consideration of safety issues associated with viral vector vaccines requires a clear understanding of the agents used for construction of the vaccine. These include (1) the wild type virus from which the vector is derived, referred to in the template as ''wild type virus"; (2) the vector itself before incorporation of the foreign antigen, referred to in the template as ''viral vector"; and (3) the final recombinant viral vector vaccine, referred to in the template as ''vaccine". Wild type viruses used as vectors may originate from human or non-human hosts and may have low or high pathogenic potential in humans regardless of species of origin. Viral vectors can originate from attenuated human vaccines, from attenuated human viruses, from human viruses with low pathogenic potential, from animal viruses with low human pathogenic potential, and from vectors (for the expression of proteins) which are then adapted as a viral vector (such as DNA plasmids or baculovirus vector vaccines) to be used as a vaccine in humans or animals. Thus, viral vectors usually, but not always, have properties in a human host that differ from wild type virus from which they were derived. Incorporation of a target antigen into a viral vector to create a vaccine may alter the properties of the vector such that the vaccine may have properties that differ from the vector. The Brighton Collaboration Vaccine Vector template is designed to describe vectors into which transgenes may be incorporated to create vaccines. However, pursuant to understanding completely the safety aspects of a given vector, consideration is given to the wild type virus from which the vector is derived ( The world was declared to be free of smallpox in May 1980 and this was because of global vaccination with vaccinia virus (VACV) based vaccines [3] . Although these vaccines were very successful at preventing smallpox there were serious adverse events indicating the need for a less virulent vaccine [4, 5] .
Due to these often-severe post-vaccination complications associated with Vaccinia viruses, there were several attempts to generate a more attenuated, safe smallpox vaccine. Modified vaccinia Ankara (MVA) originates from the dermal Vaccinia Virus Ankara strain (Chorioallantois Vaccinia Virus Ankara, CVA) that was maintained in the Vaccination Institute Ankara for many years and used as the basis for vaccination of humans. During the period of 1960 to 1974, Prof. Anton Mayr and his colleagues (University of Munich, Germany, Institute for Microbiology and Infectious Diseases of Animals) succeeded in attenuating CVA by over 570 continuous passages in primary CEF (chicken embryo fibroblast) cells.
A reduced virulence of CVA was reported from passage 371 on CEF cells [6] . From passage 516, the attenuated CVA virus was renamed MVA to discriminate it from other attenuated Vaccinia virus strains [7] [8] [9] . In clinical trials with MVA, the pock lesions associated with vaccinia virus vaccination are not seen [9] . This attenuated MVA vaccine was used in more than 120,000 vaccinees for priming prior to administration of a conventional smallpox vaccine in a two-step protocol used in the 1970s in Europe [5, 8, 10] .
In the last decades, multiple recombinant MVA vectors have been tested as vaccine candidates against various pathogens, such as human immunodeficiency viruses, Mycobacterium tuberculosis, Plasmodium falciparum or Middle East Respiratory Syndrome virus [8, 11] .
MVA-BN, that is derived from the MVA strain developed in Prof. Anton Mayr's laboratory, is a further attenuated MVA strain, which has lost its ability to replicate in most mammalian cell types, including human cell lines and is safe in severely immune compromised animals [12, 13] . The hallmark of MVA-BN is the fact that it does not productively replicate in the human keratinocyte cell line HaCat, the human cervix adenocarcinoma cell line HeLa, the human embryo kidney cell line 293 (HEK293), and the human bone osteosarcoma cell line 143B [12, 14] .
However, like other MVA strains, MVA-BN effectively infects mammalian cells. Infection of mammalian cells results in transcription of the viral genes, but no MVA-BN virus is released from the cells due to a genetic block in the viral assembly and egress. The infected cells eventually undergo apoptosis (programmed cell death) [15] [16] [17] . There are several deletions and other mutations in MVA that account for the change in host-range of the virus. Six major deletions mainly account for a reduction in the size of the original vaccinia genome from 204.5 kb to 178 kb for the MVA strain [14, 18] . Sequencing of the genome revealed that these deletions included immune evasion genes, host interactive protein genes and some structural proteins [19] .
Due to the lack of replication competence in many mammalian cells including human cells, MVA-BN can be safely administered to immunocompromised humans. This safety feature has also been confirmed in severely immunocompromised animals [12, 13] . MVA-BN is now a licensed smallpox vaccine (since 2013 in EU and Canada and since 2019 in the US, where it is also licensed as MVA-BN was shown to be more attenuated compared to two other MVA isolates, namely MVA-572 and MVA-I721, and even fails to replicate in immune compromised animals [10, 12] . Despite its high attenuation and reduced virulence, MVA-BN has been shown to elicit both humoral and cellular immune responses to Vaccinia virus and foreign genes cloned into the MVA-BN genome [31] [32] [33] [34] [35] [36] . MVA is a potent inducer of type I interferon (IFN) in human cells. MVA expresses a soluble interleukin-1 receptor, which has been implicated as an antivirulence factor for certain Poxviruses. MVA does not express soluble receptors for IFN-c, IFN-a/-b, tumor necrosis factor and CC chemokines [37] . Neurovirulence assessment of vaccinia virus based smallpox vaccines demonstrated the inability of MVA to replicate in suckling mouse brains following intracranial inoculation of 10-100 plaque forming units (pfu) of virus, while all other vaccinia virus strains tested (Dryvax Ò , Lister, Copenhagen, IHD-J, WR) replicated to peak titers of 10 7 to 10 8 pfu per gram tissue. Moreover, none of the doses of MVA tested, i.e. up to 10 5 pfu administered intracranially, induced death in the suckling mice. In contrast, mortality induced by Dryvax Ò started at a dose of 10 pfu; Lister, Copenhagen, IHD-J and WR induced death in some mice at 1 pfu and injection of 10 3 pfu was 100% lethal confirming neurovirulence reported for these strains [38] . MVA-BN replicates extensively and rapidly in CEF cells and also in certain other avian cell lines.
(replicating or non-replicating)
MVA-BN is a non-replicating vector in humans. VACV is the virus used for the replicating smallpox vaccine that was utilized during eradication and now ACAM2000.
[39]
3.2 What is the natural host for the wild type virus?
The original host is unknown, but VACV can replicate in a range of animals including primates, rodents, lagomorphs and ungulates as well as humans.
The origin of the VACV is debated and there is some evidence that it originated from a horse poxvirus which was able to infect cows. In Brazil and in India VACV is endemic in animals, with occasional transmission to humans, and is thought to originate from smallpox vaccine campaigns.
[ [39] [40] [41] [42] [43] 3.3. How is the wild type virus normally transmitted?
The typical manifestation of the wildtype virus infection are vesiculopustular lesions or dermal vesicles (pox lesions). These lesions contain infectious virus particles. Transmission can occur by close contact with infected area. There is no evidence that VACV is transmitted via airborne infection.
There is evidence that shedding of VACV from the vaccination lesion of healthy primary vaccinees occurs from about the third day to the end of the third week after vaccination. There are rare reports of transmission of VACV.
[44]
3.4. Does the wild type virus establish a latent or persistent infection?
No, the infections are acute [45] 3.5. Does the wild type virus replicate in the nucleus?
No. Poxviruses replicate in the cytoplasm.
[46]
(continued on next page) [ 51, 52] Reference for myocarditis and/or pericarditis frequency: [53] In immunocompromised humans Can be fatal and so vaccination with VACV is contraindicated.
Applicable for the replicating vacciniabased smallpox vaccines [54, 55] In human neonates, infants, children Children <12 months of age have an increased rate of the complications listed above for healthy human host [55, 56] During pregnancy and in the unborn in humans 
Poxviruses make excellent vaccine delivery vehicles since their genomes allow large insertions of foreign DNA [20, 21] . Conventionally, foreign genes are inserted into poxviruses by homologous recombination into non-essential genes or into intergenic regions [22] . The genes are under the control of a poxvirus promoter and may have a reporter gene or selection marker to aid selection of recombinants [23] [24] [25] [26] . The foreign genes are usually modified to remove the poxvirus early transcription termination signals (TTTTTNT) [27] and must be devoid of introns. Recently a Horsepox virus genome has been made by chemical synthesis and rescued by coinfection with Shope fibroma virus [28] demonstrating that this strategy can potentially be used in the future to synthesize other poxviruses. One of the most successful poxvirus vectored vaccines is the VACV vectored rabies vaccine distributed in oral baits for foxes, which has almost completely eradicated terrestrial rabies in parts of Europe [29, 30] . Host restricted poxviruses, such as the canary poxvirus, ALVAC, have been registered as commercial vaccine vectors for a number of veterinary diseases including equine influenza, canine distemper, rabies, feline leukemia and West-Nile fever [31] .
This publication presents the properties of MVA-BN as a vaccine vector and specifically focuses on MVA-BN-Filo as a component of an Ebola vaccine two-dose regimen (Ad26.ZEBOV/MVA-BN-Filo) which was granted Marketing Authorization by the European Commission on July 1, 2020 [32] (Table 1 ).
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.
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors Ariane Volkmann, Heinz Weidenthaler, and Thomas Meyer are employees of Bavarian Nordic.