Vaccines based on replication incompetent Ad26 viral vectors: Standardized template with key considerations for a risk/benefit assessment

Review Vaccines based on replication incompetent Ad26 viral vectors: Standardized template with key considerations for a risk/benefit assessment q Jerome Custers a , Denny Kim b , Maarten Leyssen a , Marc Gurwith c , Frank Tomaka b , James Robertson d , Esther Heijnen a , Richard Condit e , Georgi Shukarev a , Dirk Heerwegh f , Roy van Heesbeen a , Hanneke Schuitemaker a , Macaya Douoguih a , Eric Evans c , Emily R. Smith c , Robert T. Chen c , For the Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) 1 a Janssen Vaccines & Prevention, Leiden, the Netherlands b Janssen Research & Development, Titusville, NJ, USA c Brighton Collaboration, A Program of the Task Force for Global Health, Decatur, GA, USA d Independent Adviser, United Kingdom e Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA f Janssen Pharmaceutica NV, Beerse, Belgium article info Article history: Received 28 August 2020 Accepted 2 September 2020 Available online 3 October 2020 Keywords: Vaccine Viral vector Safety Benefit/risk Replication-incompetent Ad26 abstract Replication-incompetent adenoviral vectors have been under investigation as a platform to carry a variety of transgenes, and express them as a basis for vaccine development. A replication-incompetent adenoviral vector based on human adenovirus type 26 (Ad26) has been evaluated in several clinical trials. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety and features of recombinant viral vector vaccines. This paper reviews features of the Ad26 vectors, including tabulation of safety and risk assessment characteristics of Ad26-based vaccines. In the Ad26 vector, deletion of E1 gene rendering the vector replication incompetent is combined with additional genetic engineering for vaccine manufacturability and transgene expression optimization. These vaccines can be manufactured in mammalian cell lines at scale providing an effective, flexible system for high-yield manufacturing. Ad26 vector vaccines have favorable thermostability profiles, compatible with vaccine supply chains. Safety data are compiled in the Ad26 vaccine safety database version 4.0, with unblinded data from 23 ongoing and completed clinical studies for 3912 participants in five different Ad26-based vaccine programs. Overall, Ad26-based vaccines have been well tolerated, with no significant safety issues identified. Evaluation of Ad26-based vaccines is continuing, with >114,000 participants vaccinated as of 4th September 2020. Extensive evaluation of immunogenicity in humans shows strong, durable humoral and cellular immune responses. Clinical trials have not revealed impact of pre-existing immunity to Ad26 on vaccine immunogenicity, even in the presence of Ad26 neutralizing antibody titers or Ad26-targeting T cell responses at baseline. The first Ad26-based vaccine, against Ebola virus, received marketing authorization from EC on 1st July 2020, as part of the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen. New developments based on Ad26 vectors are underway, including a COVID-19 vaccine, which is currently in phase 3 of clinical evaluation. Ó2020 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/). https://doi.org/10.1016/j.vaccine.2020.09.018 0264-410X/Ó2020 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/). q Purpose of document: Risk benefit assessment of the Ad26 replication-incompetent viral vector using the Brighton Collaboration V3SWG Standardized Template for Collection of Key Information for Risk Assessment of Viral Vaccine Vector Candidates. 1 See Acknowledgement for other V3SWG members. Vaccine 39 (2021) 3081–3101 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Contents 1. Introduction . . . ..................................................................................................... 3082 2. Background. . . . ..................................................................................................... 3082 2.1. Ad26 vector development........................................................................................ 3082 2.2. Manufacturing, formulation and stability . . . ........................................................................ 3082 2.3. Safety, tolerability and immunogenicity of Ad26-based vaccines in humans . . . . . . . . . . . . . .................................. 3083 3. Disclaimer. . . . . ..................................................................................................... 3099 4. Purpose of document . . . . . . . . . . . . . . . .................................................................................. 3099 Declaration of Competing Interest . . . . .................................................................................. 3099 Acknowledgements . . . . . . . . . . . . ...................................................................................... 3099 Authorship. . . . . . ..................................................................................................... 3099 References . . . . ..................................................................................................... 3099 1. Introduction The Brighton Collaboration (www.brightoncollaboration.org) V3SWG is an established collaboration who aim to enhance the science of vaccine safety research (http://cms.brightoncollaboration.org: 8080/public/what-we-do/setting-standards/case-defini tions/process). The Brighton V3SWG uses a standardized template to describe the key characteristics of novel vaccine vectors, compiled from the latest research, to facilitate scientific discourse among key stakeholders [1]. 2. Background The adenovirus type 26 (Ad26) wild type virus was first isolated in 1956 from an anal specimen of a 9-month-old male child [2].As described in that study, 4 different isolates were obtained from anal and throat swabs from different children, some of whom experienced mild self-limiting enteric infections. Although, none of the illnesses could etiologically be associated with the isolated adenoviruses, it suggests that wild type Ad26 can, presumably, cause asymptomatic or minor illness [2]. Human Ad26 has been considered to be a low-prevalent adenovirus due to the low frequency of Ad26 neutralizing antibodies in various populations compared with human adenovirus type 5. For example, a seroprevalence study of the 51 human adenovirus serotypes known at the time showed that several serotypes from particularly subgroups B and D, including Ad26, were rare in a Belgian population [3], suggesting that vectors (rAd) derived from these serotypes might be useful alternatives to Ad5-based vectors for vaccine development, since for Ad5-based vectors it was shown that their high prevalence hampered their clinical use [4–8]. More extensive seroprevalence and immunogenicity studies showed that while all of these vectors exhibited low seroprevalence, Ad26-based vaccine candidates were the most immunogenic in animals [9]. Further studies have shown that, depending on geographical location, 10%–90% of people tested have neutralizing antibodies against Ad26. However, neutralization titers are low to intermediate compared with those observed for other adenovirus types [10–13]. 2.1. Ad26 vector development Adenovirus genomes are linear, non-segmented doublestranded DNA molecules with inverted terminal repeat (ITR) sequences at each end. The vector system for replicationincompetent Ad26 vaccine vectors consists of an adaptor plasmid and a cosmid [9]. The adaptor plasmid contains the left end of the genome containing the left ITR and the packaging signal. It also contains a transgene expression cassette in place of the E1 region and a 2.5 kb fragment downstream of the E1 region to enable homologous recombination with the cosmid. The cosmid contains the majority of the Ad genome, spanning from the pIX sequence to the right ITR, with a deletion of the E3 region and a modified E4 open reading frame 6 (E4orf6). Transfection into a suitable packaging cell line (HEK293 cells, PER.C6 Ò cells) and subsequent homologous recombination of the adaptor plasmid and cosmid results in the generation of a replication-incompetent E1/E3-deleted Ad26 vector. Packaging cell lines like the HEK293 and PER.C6 Ò cell lines contain the E1 region of adenovirus serotype 5 (Ad5). Because within the adenoviral life cycle, E1 protein 55 K and E4 protein Orf6 form a complex that is pivotal for high-level late-gene expression, the E4-Orf6 sequence of Ad26 is replaced by the corresponding sequence from Ad5 in the vector. This modification has previously been shown to be necessary to allow for the efficient production of rAd35 virus on Ad5 E1-complementing cells [14]. Finally, compensation for the loss of E3 is not needed since the E3 proteins are not essential for adenoviral growth in vitro but are involved in down regulating cellular immunological response mechanisms in an attempt of the adenovirus to escape the host immune system [15]. Most adenovirus serotypes use the coxsackievirus-adenovirus receptor for attachment to the target cell [16,17]. In contrast, Ad26 has been reported to utilize CD46 as its primary cellular receptor [9,18], but more recent reports indicated only a limited interaction between Ad26 and CD46, even showing evidence of a role for a vb3 integrin for efficient transduction of epithelial cells, or interaction with sialic acid [19,20]. These data suggest that receptor usage by Ad26 might be host cell-type dependent in vitro [19,20]. Target cells in vivo in the natural host are not known, but Ad26 virus can infect a variety of cell types in vitro. Detailed studies dissecting the attachment, internalization and intracellular trafficking of adenoviral vectors have shown that Ad26, amongst others, accumulate in the late endosome to a larger extent and trigger innate immune pathways differentially compared with Ad5-based vectors [21]. Whether and how these differences may translate into differential profiles of adaptive immunity against the vaccine antigen is not known. 2.2. Manufacturing, formulation and stability Ad26 vector-based vaccines are manufactured using the E1complementing PER.C6 Ò cell line, a continuous, human cell line capable of supporting the manufacturing of replication incompetent adenoviral vectors [22]. One of the key strengths of this cell platform is that the cells can grow in suspension in serum-free media to very high cell densities. Cell counts of 100 million cells/ mL, with a high percentage of viable cells, can be reached within 10 days of cell culture. Janssen has taken advantage of the ability to grow the PER.C6 Ò cell line at high cell densities in a so called ‘‘intensified process”. Cell-specific yields are in the same range as is J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3082 generally achieved with other Adenoviral vectors and E1complementing cell lines, therefore, due to the higher cell densities yields per volume unit are higher. The complete manufacturing process has now been scaled up to 1,000 L allowing manufacturing at a commercial scale. While lyophilized vaccines are generally more heat-stable than non-lyophilized alternatives [23], liquid vaccine formulations may have several advantages over lyophilized vaccines, including ease of manufacture, packaging, and simple administration procedures [24]. For Ad26-based vectors, progress in formulation development has allowed for long-term storage of product at 2–8 °C, enabling product distribution through existing vaccine supply chains. Assessments of robustness during storage, handling and distribution conditions have shown that recombinant Ad26 vectors can be maintained stable under frozen conditions or at 2–8 °C, and, furthermore, showed to be stable in-use with a syringe/needle, also when subjected to agitation or temperature excursions [25]. 2.3. Safety, tolerability and immunogenicity of Ad26-based vaccines in humans Ad26-based vaccines have been extensively tested in completed and ongoing clinical studies from multiple clinical programs (Fig. 1). The following vaccine candidates have been evaluated in clinical programs: Ad26.ZEBOV for Ebola virus Ad26.Mos.HIV, Ad26.Mos4.HIV, and Ad26.ENVA.01 for HIV Ad26.CS.01 for Malaria Ad26.RSV.FA2 and Ad26.RSV.preF for RSV Ad26.Filo for Filovirus Ad26.ZIKV.001 for Zika Ad26.HPV16 and Ad26.HPV18 for HPV The adenoviral vaccine (AdVac) safety database report version 4.0 (14 May 2019) contains data from 23 clinical studies, for a total of 3912 study participants, using the following Ad26-based vaccines: Ad26.ZEBOV (Ebola program; 9 studies [26,27];Table 1), Ad26.ENVA.01, Ad26.Mos.HIV, and Ad26.Mos4.HIV (HIV program; 8 studies [28–32];Table 2), Ad26.CS.01 (Malaria program; 1 study [33];Table 3), Ad26.RSV.FA2 and Ad26.RSV.preF (RSV program; 4 studies; Table 4), and Ad26.Filo (Filovirus program; 1 study; Table 5). A full overview of the clinical studies can be found in Tables 1–5. The cut-off date for this report was 21 December 2018. Data from all unblinded studies for which the clinical database had been locked and unblinded, either for interim, primary or final analyses, are included in this review (Table 6,[2,9–13, 18–20,34–71]). Extensive safety and immunogenicity data have been obtained from these studies. Overall, in our analysis, all Ad26-based vaccines have been well tolerated, with no significant safety issues identified from the data currently available in the AdVac safety database. Post-vaccination reactogenicity after administration of Ad26-based vaccines consisted of mild-to-moderate adverse events (AEs) that started 1–2 days after vaccination and resolved within 1–3 days. Most participants received Ad26 dose of 5 10 10 viral particles (vp) but relatively few participants received doses of 1 10 9 vp, 110 10 vp, 9 10 10 vp, and 1 10 11 vp making it difficult to establish an effect of dose level on reactogenicity. When only dose groups with more than 100 participants were considered, there was a trend towards an increase in the incidence of local and systemic reactogenicity with an increase in Ad26 dose. Overall, the proportions of participants reporting unsolicited AEs, as well as serious AEs were comparable between the active vaccine and placebo groups. As expected with most vaccines, pyrexia was observed more frequently after administration of Ad26-based vaccines, compared with placebo, and occurred most frequently in younger children. Most episodes of pyrexia were mild or moderate in severity. Occurrences of severe pyrexia were observed with similar frequency after Ad26-based vaccine or placebo administration. As of 4th September 2020, Ad26-based vaccines have been administered to more than 114,000 individuals and have been shown to have an acceptable safety profile and induce strong and durable humoral and cellular immune responses in clinical trials (Fig. 1). The Ebola vaccine program has largely contributed to this expansion of the experience on Ad26-based vaccines, with two ongoing projects: a large clinical study in the Democratic Republic of the Congo [72] and a government-led vaccination program in Rwanda after conditional approval under exceptional emergency [73]. The Ebola vaccine regimen, consisting of two components, Zabdeno (Ad26.ZEBOV) and Mvabea (MVA-BN-Filo), given approximately 8 weeks apart, has received EC marketing authorization on 1st July 2020 [74]. Ad26-based vaccines are capable of inducing antibodies that have neutralizing and/or other effector functions. The induction of strong CD4+ and CD8+ T cell responses has been correlated with protection or functional cure in pre-clinical models [32,52,53]. Responses induced by Ad26-based vaccines can potentially be further augmented and sustained for even longer periods in combination vaccination regimens with, for example, the same antigens in a different presentation format. Combinations with other antigen presentations have been evaluated with soluble proteins, which provided enhanced immune response, or with transgenes in other viral vectors, for instance, the Modified Vaccinia Ankara (MVA) vector, to further expand the humoral and cellular responses. For Ad5-based vectors, vector-targeting immune responses have been shown to decrease the immune responses against the Fig. 1. Summary of number of Ad26-based vaccines administered (full experience and AdVac Safety Database). J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3083 Table 1 Summary of Ad26 Ebola vaccine clinical studies. Study Sponsor (IND holder) Vaccine Status Phase/Blind/Allocation/Participants/ Country Protocol-specified vaccination groups Planned number of participants Reference NCT No. VAC52150 EBL1001 Janssen Ad26.ZEBOV, MVA-BN-Filo Completed Phase 1/1st part of the study/ Double-blind (observer-blind)/ Randomized/Healthy adults/UK 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 29) 15 NCT02313077 [26,27] 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 57) 15 510 10 vp Ad26.ZEBOV (Day 1) 110 8 TCID 50 MVA-BN-Filo (Day 29) 15 510 10 vp Ad26.ZEBOV (Day 1) 110 8 TCID 50 MVA-BN-Filo (Day 57) 15 Placebo 12 2nd part of the study/Open-label/ Non-randomized/Healthy adults/UK 510 10 vp Ad26.ZEBOV (Day 1) 110 8 TCID 50 MVA-BN-Filo (Day 15) 15 VAC52150 EBL1002 Janssen Ad26.ZEBOV, MVA-BN-Filo Completed Phase 1/Double-blind (observer-blind)/Randomized/ Healthy adults/US 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 15) 19 NCT02325050 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 15) 510 10 vp Ad26.ZEBOV (Day 360) 11 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 29) 4 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 29) 510 10 vp Ad26.ZEBOV (Day 360) 11 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 57) 3 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 57) 510 10 vp Ad26.ZEBOV (Day 360) 12 510 10 vp Ad26.ZEBOV (Day 1) 110 8 TCID 50 MVA-BN-Filo (Day 29) 2 510 10 vp Ad26.ZEBOV (Day 1) 110 8 TCID 50 MVA-BN-Filo (Day 29) 510 10 vp Ad26.ZEBOV (Day 360) 13 110 8 TCID 50 MVA-BN-Filo (Day 1) 110 8 TCID 50 MVA-BN-Filo (Day 15) 510 10 vp Ad26.ZEBOV (Day 360) 7 510 10 vp Ad26.ZEBOV (Day 1) 510 10 vp Ad26.ZEBOV (Day 15) 2 510 10 vp Ad26.ZEBOV (Day 1) 510 10 vp Ad26.ZEBOV (Day 15) 110 8 TCID 50 MVA-BN-Filo (Day 360) 7 510 10 vp Ad26.ZEBOV (Day 1) 4.4 10 8 TCID 50 MVA-BN-Filo (Day 15) 4 510 10 vp Ad26.ZEBOV (Day 1) 4.4 10 8 TCID 50 MVA-BN-Filo (Day 15) 510 10 vp Ad26.ZEBOV (Day 360) 11 110 11 vp Ad26.ZEBOV (Day 1) 4.4 10 8 TCID 50 MVA-BN-Filo (Day 29) 5 110 11 vp Ad26.ZEBOV (Day 1) 4.4 10 8 TCID 50 MVA-BN-Filo (Day 29) 110 11 vp Ad26.ZEBOV (Day 360) 10 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 8) 15 Placebo 25 J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3084 VAC52150 EBL1003 Janssen Ad26.ZEBOV, MVA-BN-Filo Completed Phase 1/Double-blind (observer-blind)/Randomized/ Healthy adults/Kenya 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 29) 15 NCT02376426 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 57) 15 510 10 vp Ad26.ZEBOV (Day 1) 110 8 TCID 50 MVA-BN-Filo (Day 29) 15 510 10 vp Ad26.ZEBOV (Day 1) 110 8 TCID 50 MVA-BN-Filo (Day 57) 15 Placebo 12 VAC52150 EBL1004 Janssen Ad26.ZEBOV, MVA-BN-Filo Completed Phase 1/Double-blind (observer-blind)/Randomized/ Healthy adults/Tanzania, Uganda 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 29) 15 NCT02376400 110 8 TCID 50 MVA-BN-Filo (Day 1) 510 10 vp Ad26.ZEBOV (Day 57) 15 510 10 vp Ad26.ZEBOV (Day 1) 110 8 TCID 50 MVA-BN-Filo (Day 29) 15 510 10 vp Ad26.ZEBOV (Day 1) 110 8 TCID 50 MVA-BN-Filo (Day 57) 15 Placebo 12 VAC52150 EBL2001 Janssen Ad26.ZEBOV, MVA-BN-Filo Completed Phase 2/Double-blind (observer-blind)/Randomized/ Healthy adults/France, UK 510 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 29) 174 NCT02416453 510 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 57) 174 510 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 85) 174 510 10 vp Ad26.ZEBOV (Day 1) 15 Placebo 93 VAC52150 EBL2002 Janssen Ad26.ZEBOV, MVA-BN-Filo Ongoing Phase 2/Double-blind (observer-blind)/Randomized/ Healthy adults 18–70 yrs; HIV+ adults 18–50 yrs; healthy children 4–17 yrs/Burkina Faso, Uganda, Côte D’Ivoire, Kenya 510 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 29) 385 NCT02564523 510 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 57) 385 510 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 85) 110 Placebo 176 VAC52150 EBL3001 Janssen Ad26.ZEBOV, MVA-BN-Filo Ongoing Phase 3/Open-label uncontrolled stage in healthy participants 18 years (Stage 1)/Double-blind randomized stage in healthy participants 1 year (Stage 2)/Sierra Leone 510 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 57) 772 NCT02509494 Placebo 244 VAC52150 EBL3002 Janssen Ad26.ZEBOV, MVA-BN-Filo Completed Phase 3/Double-blind (observer-blind)/Randomized/ Healthy adults/US 0.8 10 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 57) 150 NCT02543567 210 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 57) 150 510 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 57) 150 Placebo 75 VAC52150 EBL3003 Janssen Ad26.ZEBOV, MVA-BN-Filo Completed Phase 3/Double-blind (observer-blind)/Randomized/ Healthy adults/US 510 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 57) 282 NCT02543268 Placebo 47 Cut-off for studies 21 December 2018. Ad26, adenovirus serotype 26; BN, Bavarian Nordic; EBOV, Ebola virus; Inf U, infectious unit; MVA, modified vaccinia virus Ankara; TCID 50 , 50% tissue culture infective dose; vp, viral particles; ZEBOV, Zaire Ebola virus. J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3085 Table 2 Summary of Ad26 HIV vaccine clinical studies. Study Sponsor (IND holder) Vaccine Status Phase/Blind/Allocation/ Participants/Country Protocol-specified vaccination groups Planned number of participants Reference NCT No. IPCAVD-001 DAIDS Ad26.ENVA.01 Completed Phase 1/Double-blind (observer-blind)/ Randomized/Healthy adults/US 110 9 vp Ad26.ENVA.01 (Weeks 0, 4, 24) 10 NCT00618605 [28,29] 110 10 vp Ad26.ENVA.01 (Weeks 0, 4, 24) 10 110 11 vp Ad26.ENVA.01 (Weeks 0, 4, 24) 10 510 10 vp Ad26.ENVA.01 (Weeks 0, 24) 10 110 10 vp Ad26.ENVA.01 (Weeks 0, 24) 10 Placebo 10 IPCAVD-003 DAIDS Ad26.ENVA.01 Completed Phase 1/Double-blind (observer-blind)/ Randomized/Healthy adults (Ad26 seronegative and Ad26 seropositive)/US 510 10 vp Ad26.ENVA.01 (Week 0) 18 NCT01103687 [30] Placebo 6 IPCAVD-004 IAVI Ad26.ENVA.01 Ad35.ENV Completed Phase 1/Double-blind (observer-blind)/ Randomized/Healthy adults/US, Kenya, Rwanda, South Africa 510 10 vp Ad26.ENVA.01 (Month 0) 510 10 vp Ad35.ENV (Month 6) 10 NCT01215149 [31] 510 10 vp Ad35.ENV (Month 0) 510 10 vp Ad26.ENVA.01 (Month 6) 10 510 10 vp Ad26.ENVA.01 (Month 0) 510 10 vp Ad35.ENV (Month 3) 42 510 10 vp Ad35.ENV (Month 0) 510 10 vp Ad26.ENVA.01 (Month 3) 42 510 10 vp Ad26.ENVA.01 (Month 0) 510 10 vp Ad26.ENVA.01 (Month 3) 32 510 10 vp Ad35.ENV (Month 0) 510 10 vp Ad35.ENV (Month 3) 32 Placebo 44 HIV-V-A004 Janssen Ad26.Mos.HIV Ongoing* Phase 1/2a/ Double-blind/ Randomized/Healthy adults/US, Thailand, Rwanda, Uganda, South Africa 510 10 vp Ad26.Mos.HIV (Weeks 0, 12) 510 10 vp Ad26.Mos.HIV + 250 mcg gp140 + adjuvant (Weeks 24, 48) 50 NCT02315703 [32] 510 10 vp Ad26.Mos.HIV (Weeks 0, 12) 510 10 vp Ad26.Mos.HIV + 50 mcg gp140 + adjuvant (Weeks 24, 48) 50 510 10 vp Ad26.Mos.HIV (Weeks 0, 12) 510 10 vp Ad26.Mos.HIV + placebo (Weeks 24, 48) 50 510 10 vp Ad26.Mos.HIV (Weeks 0, 12) 10 8 pfu MVA-Mosaic + 250 mcg gp140 + adjuvant (Weeks 24, 48) 50 510 10 vp Ad26.Mos.HIV (Weeks 0, 12) 10 8 pfu MVA-Mosaic + 50 mcg gp140 + adjuvant (Weeks 24, 48) 50 510 10 vp Ad26.Mos.HIV (Weeks 0, 12) 10 8 pfu MVA-Mosaic + placebo (Weeks 24, 48) 50 510 10 vp Ad26.Mos.HIV (Weeks 0, 12) 250 mcg gp140 + adjuvant + placebo (Weeks 24, 48) 50 Placebo (Weeks 0, 12, 24, 48) 50 VAC89220 HPX1002 Janssen Ad26.Mos.HIV OngoingyPhase 1/Double-blind/ Randomized/Healthy adults/US 510 10 vp Ad26.Mos.HIV (Weeks 0, 12) 510 10 vp Ad26.Mos.HIV + 250 mcg Clade C gp140 + adjuvant (Weeks 24, 48) 10 NCT02685020 510 10 vp Ad26.Mos.HIV + 250 mcg Clade C gp140 + adjuvant (Weeks 0, 12, 24) 10 J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3086 Table 2 (continued) Study Sponsor (IND holder) Vaccine Status Phase/Blind/Allocation/ Participants/Country Protocol-specified vaccination groups Planned number of participants Reference NCT No. 510 10 vp Ad26.Mos.HIV (Week 0) 510 10 vp Ad26.Mos.HIV + 250 mcg Clade C gp140 + adjuvant (Week 8, 24) 10 Placebo 6 VAC89220HPX2003 Janssen Ad26.Mos4.HIV OngoingàPhase 1/2a/ Double-blind/ Randomized/Healthy adults/US, Kenya, Rwanda 510 10 vp Ad26.Mos4.HIV (Weeks 0, 12) 510 10 vp Ad26.Mos.HIV + 250 mcg Clade C gp140 + adjuvant (Weeks 24, 48) 25 NCT02935686 510 10 vp Ad26.Mos4.HIV (Weeks 0, 12) 510 10 vp Ad26.Mos.HIV + 250 mcg Clade C + Mosaic gp140 + adjuvant (Weeks 24, 48) 100 Placebo 25 VAC89220HPX2004 Janssen Ad26.Mos.HIV Ad26.Mos4.HIV Ongoing§ Phase 1/2a/ Double-blind/ Randomized/Healthy adults/US, Rwanda 510 10 vp Ad26.Mos.HIV (Weeks 0, 12) 510 10 vp Ad26.Mos.HIV + 250 mcg Clade C gp140 + adjuvant (Weeks 24, 48) 55 NCT02788045 510 10 vp Ad26.Mos4.HIV (Weeks 0, 12) 510 10 vp Ad26.Mos4.HIV + 250 mcg Clade C gp140 + adjuvant (Weeks 24, 48) 110 Placebo 33 VAC89220HTX1001 Janssen Ad26.Mos.HIV Completed–Phase 1/2a/ Double-blind/ Randomized/HIV-1 infected adults/ Thailand 510 10 vp Ad26.Mos.HIV (Weeks 0, 12) 110 8 pfu MVA-Mosaic (Weeks 24, 48) 18 NCT02919306 Placebo 9 Cut-off for studies 21 December 2018. * The main study has been completed; the long-term extension phase is still ongoing. Week 96 Final Analysis has been used in AdVac Safety Database. yWeek 72 Final Analysis has been used in AdVac Safety Database. àWeek 52 Interim Analysis was used in AdVac Safety Database. § The main study has been completed; the long-term extension phase is still ongoing. Week 72 Final Analysis has been used in AdVac Safety Database. –Week 96 Final Analysis has been used in AdVac Safety Database. Ad26, adenovirus serotype 26; Ad35, adenovirus serotype 35; Enva, envelope A; gp, glycoprotein; mcg, microgram; Mos, mosaic; MVA, modified vaccinia Ankara; pfu, plaque-forming units; vp, viral particles. J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3087 Table 3 Summary of Ad26 Malaria vaccine clinical studies. Study Sponsor (IND holder) Vaccine Status Phase/Blind/Allocation/ Participants/Country Protocol-specified vaccination groups Planned number of participants Reference NCT No. MAL-V-A001 Janssen Ad35.CS.01 Ad26.CS.01 Completed Phase 1/2a/ Double-blind (observer-blind)/ Randomized/Healthy adults/US 110 10 vp Ad35.CS.01 (Days 0, 28), 110 10 vp Ad26.CS.01 (Day 55) 10 NCT01397227 [33] 510 10 vp Ad35.CS.01 (Days 0, 28), 510 10 vp Ad26.CS.01 (Day 55) 20 Placebo 6 Cut-off for studies 21 December 2018. Ad26, adenovirus serotype 26; Ad35, adenovirus serotype 35; CS, circumsporozoite; vp, viral particles. Table 4 Summary of Ad26 RSV vaccine clinical studies. Study Sponsor (IND holder) Vaccine Status Phase/Blind/Allocation/ Participants/Country Protocol-specified vaccination groups Planned number of participants Reference NCT No. VAC18192 RSV1001 Janssen Ad35.RSV. FA2 Ad26.RSV. FA2 Completed Phase 1/Double-blind (observer-blind)/ Randomized/Healthy adults/US 110 11 vp Ad35.RSV.FA2 (Days 1, 85), 510 10 vp Ad26.RSV.FA2 (Day 169) 12 NCT02440035 110 11 vp Ad35.RSV.FA2 (Days 1, 169), placebo (Day 85) 12 110 11 vp Ad35.RSV.FA2 (Day 1), placebo (Day 85), 510 10 vp Ad26.RSV.FA2 (Day 169) 12 Placebo (Days 1, 85), 510 10 vp Ad26.RSV.FA2 (Day 169) 12 VAC18192 RSV1003 Janssen Ad35.RSV. FA2 Ad26.RSV. FA2 Completed Phase 1/Double-blind (observer-blind)/ Randomized/Healthy adults/US 510 10 vp Ad26.RSV.FA2 (Days 1, 85), 110 11 vp Ad35.RSV.FA2 (Day 169) 12 NCT02561871 510 10 vp Ad26.RSV.FA2 (Day 1), 110 11 vp Ad35.RSV.FA2 (Day 85), placebo (Day 169) 12 Placebo 8 VAC18193 RSV2002 Janssen Ad26.RSV. preF Ongoing Phase 2a/Double-blind (observer-blind)/ Randomized/Healthy adults/UK 110 11 vp Ad26.RSV.preF (Day 28) Challenged intranasally with RSV-A at Day 0 22 NCT03334695 Placebo (Day 28) Challenged intranasally with RSV-A at Day 0 22 VAC18193 RSV2003 Janssen Ad26.RSV. preF Completed Phase 2a/Double-blind (observer-blind)/ Randomized/Healthy older adults (60 years of age)/US 110 11 vp Ad26.RSV.preF (Day 1), Fluarix Ò Quadrivalent (Day 1), placebo (Day 29) 90 NCT03339713 Fluarix Ò Quadrivalent (Day 1), placebo (Day 1), 110 11 vp Ad26.RSV.preF (Day 29) 90 Cut-off for studies 21 December 2018. Ad26, adenovirus serotype 26; Ad35, adenovirus serotype 35; FA2, F protein of RSV strain A2; preF, prefusion F protein; RSV, respiratory syncytial virus; vp, viral particles. Table 5 Summary of Ad26 Filovirus vaccine clinical studies. Study Sponsor (IND holder) Vaccine Status Phase/Blind/Allocation/ Participants/Country Protocol-specified vaccination groups Planned number of participants Reference NCT No. VAC69120FLV1001 Janssen Ad26.Filo Ad26.ZEBOV Completed Phase 1/Double-blind (observer-blind for subset in Group 3 who received the booster vaccination)/ Randomized/Healthy adults/US 910 10 vp Ad26.Filo (Day 1) 510 8 Inf U MVA-BN-Filo (Day 57) 15 NCT02860650 510 8 Inf U MVA-BN-Filo (Day 1) 910 10 vp Ad26.Filo (Day 57) 15 510 8 Inf U MVA-BN-Filo (Day 1) 910 10 vp Ad26.Filo (Day 15) 910 10 vp Ad26.Filo (Day 92) 15 510 10 vp Ad26.ZEBOV (Day 1) 110 8 Inf U MVA-BN-Filo (Day 57) 15 Placebo 12 Cut-off for studies 21 December 2018. Ad26, adenovirus serotype 26; BN, Bavarian Nordic; Inf U, infectious unit; MVA, modified vaccinia Ankara; vp, viral particles; ZEBOV, Zaire Ebola Virus. J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3088 Table 6 Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) standardized template V2.0 for collection of key information for risk assessment of viral vaccine vector candidates filled with Ad26 data. 1. Authorship Information 1.1. Author(s) 1.2. Date completed/updated 21 November 2019 2. Basic vector information Information 2.1. Vector name Ad26 2.2. Vector origin Family/Genus/ Species/subtype Family adenoviridae subgroup D, Human Adenovirus type 26 2.3. Vector replication in humans (replicating or non-replicating) Replication incompetent 3. Characteristics of the wild type virus from which the vector is derived Information Comments/Concerns Reference(s) 3.1. Name of wild type virus (common name; Family/Genus/Species/subtype) Family adenoviridae subgroup D, human Adenovirus type 26 3.2. What is the natural host for the wild type virus? Humans. No evidence that Adenovirus type 26 can infect other non-human animals 3.3. How is the wild type virus normally transmitted? Not known In general, human adenoviruses can be transmitted via the oral/faecal route, through aerosols and human-to-human contact. [34,35] 3.4. Does the wild type virus establish a latent or persistent infection? Not known for Adenovirus type 26 In general, Adenovirus infections are selflimiting. In some cases, adenoviruses can establish a persistent infection and can reside in certain tissues for longer periods of time. Also, prolonged shedding of infectious virus has been observed for certain adenovirus types. This is observed more prominent in immunocompromised individuals. [34,35] 3.5. Does the wild type virus replicate in the nucleus? Yes 3.6. What is the risk of integration into the human genome? Negligible Adenoviruses are considered nonintegrating according to the EMA ‘Guideline on nonclinical testing for inadvertent germline transmission of gene transfer vectors’, because they lack the machinery to actively integrate their genome into the host chromosomes. The adenoviral genome remains epichromosomal, thus avoiding the risk of integration of the viral DNA into the host genome following cell infection. Therefore, chromosomal integration of genetic material of Ad26 in the human host is unlikely. [36–39] 3.7. List any disease manifestations caused by the wild type virus, the strength of evidence, severity, and duration of disease for the following categories: In the healthy natural host Unknown for Ad26 See below In healthy human host Unknown for Ad26 In a challenge study in humans, Kasel et al. reported symptomatic infections of Ad26 wild type in human participants upon inoculation in the conjunctival sac or intranasally. Participants inoculated in the conjunctival sac developed a moderate but self-limiting conjunctivitis with positive virus isolations from the eye in the first week after infection but not thereafter. Symptoms were milder in the presence of pre-existing Ad26 antibodies. Participants who were inoculated intranasally developed barely perceptible rhinitis without associated symptoms or signs. No eye disease occurred after nasal inoculation. Shedding upon intranasal inoculation was not studied. Adenovirus type 26 was isolated from the rectum of participants who were inoculated in the conjunctival sac for up to 48 days after initial inoculation albeit without causing any gastrointestinal or systemic illness. Prolonged isolation of the virus from the gastrointestinal tract could indicate infection of cells in the gastrointestinal [40,41] (continued on next page) J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3089 HIV program Ad26.ENVA.01: 206 Ad26.Mos.HIV: 446 Ad26.Mos4.HIV: 236 Malaria program Ad26.CS.01: 28 RSV program Ad26.RSV.FA2: 59 Ad26.RSV.preF: 211 Filovirus program Ad26.Filo: 43 In more than 40 clinical studies, either ongoing or completed, with a last update as of 4 September 2020 and a vaccination campaign in Rwanda, more than 114,000 participants (including >40,000 children) were vaccinated with an Ad26-based vaccine (cut-off: 4 September 2020; estimate based on the study randomization ratios). 7.2. Method(s) used for safety monitoring: Serious AEs Yes In most studies, serious AEs and deaths were collected and reported throughout the study or up to 6 months postvaccination, regardless of time to onset. Solicited AEs Yes In most studies, solicited AEs were generally collected for a 7-day postvaccination period, using a study participant diary. Unsolicited AEs Yes Unsolicited AEs were generally collected up to 28–30 days/4 weeks postvaccination in most studies. Other active surveillance Yes Ebola/Filovirus program: A list of neuroinflammatory disorders were categorized as Immediate Reportable Events (IREs); these were reported to the sponsor within 24 h of becoming aware of the event, using the IRE Form. HIV Program: Confirmed HIV infections and potential immune-mediated diseases were considered an adverse event of special interest (AESI). All AESIs were reported to the sponsor immediately and aggregate analyses performed either at the end of study and possible at interim time points during the studies (for non-efficacy studies only). RSV Pediatric Program (not included in this review): Monitoring for severe lower respiratory tract infection (LRTIs) took place during the whole study period for paediatric participants (enhanced respiratory disease risk [ERD] surveillance). ZIKV Program (not included in this review): A list of neuroinflammatory disorders associated with ZIKV infection were categorized as IRE’s and had to be reported to the sponsor within 24 h after becoming aware of the event using the IRE Form. No IRE’s were reported. 7.3. What criteria were used for grading the AEs? 2007 US FDA Guidance for Industry Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials Yes Modified version including company standards for terms, definitions, and grading of solicited AEs. Other modifications are footnoted. The Division of AIDS Table for Grading the Severity of Adult and Pediatric AEs (version 1.0, December 2004; Clarification August 2009 and Version 2.0, November 2014) Yes Modified version including company standards for terms, definitions, and grading of solicited AEs. Other modifications are footnoted. Division of Microbiology and Infectious Diseases (DMID) Toxicity Table for use in trials enrolling healthy adults (2007 and 2014) Yes Modified version including company standards for terms, definitions, and grading of solicited AEs. Other modifications are footnoted. J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3096 Division of Microbiology and Infectious Diseases (DMID) Pediatric Toxicity Tables for Children Greater Than 3 Months of Age (2007) Yes Modified version including company standards for terms, definitions, and grading of solicited AEs. Other modifications are footnoted. 7.4. List and provide frequency of any related or possibly related serious* AEs observed: Three suspected unexpected serious adverse reactions (SUSARs) have been reported since the beginning of the Ad26 vaccines programs, as follows: Ad26.Mos.HIV (HIV-V-A004 study): - Hypersensitivity; verbatim: severe allergic response Ad26.ZEBOV (VAC52150EBL2001): - Small fiber neuropathy Ad26.Mos4.HIV (VAC89220HPX2004): - Rheumatoid arthritis N = 1 per each reported SUSAR. [32,70] 7.5. List and provide frequency of any statistically significantly increased AE or lab abnormality in vaccinee vs. control group: The safety in Ad26 participants is compared versus placebo participants. All data in the Janssen AdVac safety database were only analyzed descriptively, no statistical testing was performed. Solicited local AEs: Overall, the most frequently reported solicited local AEs were injection-site pain (58.8% of Ad26 participants, compared with 21.9% of placebo participants), injection-site warmth (20.4% and 9.9%, respectively), and injection-site swelling (10.1% and 4.8%, respectively). Injectionsite erythema, induration, and pruritus were experienced by fewer than 10% of participants. Solicited systemic AEs: Overall, the most frequently reported solicited systemic AEs for Ad26 participants (reported in 30% of participants) were malaise (53.8%), fatigue (49.0%), headache (46.2%), and myalgia (38.9%), all of which were more frequent for Ad26 participants compared with placebo (42.4%, 28.0%, 26.4%, and 16.3% of placebo participants, respectively). Pyrexia was also more frequent in Ad26 participants than in placebo participants (10.7% and 3.2%, respectively). Unsolicited AEs: For Ad26, the most frequently experienced unsolicited AEs were upper respiratory tract infection (4.9%), malaria (3.7%), headache (2.9%), and neutropenia (2.0%). For placebo, these percentages were 6.4%, 1.4%, 2.4%, and 1.6%, respectively. Clinical laboratory evaluation: Decrease in haemoglobin from baseline was the most frequent laboratory abnormality (1,408 of 2,178 Ad26 participants [64.6%] and 235 of 414 placebo participants [56.8%]). It should however be noted that, according to the FDA Toxicity Grading Scale, any decrease in hemoglobin from baseline is considered a laboratory abnormality, even if it is within the normal laboratory reference ranges. Grade 3 abnormalities were infrequent (reported in fewer than 3% of participants) and comparable between Ad26 and placebo participants. From the Janssen AdVac safety database (version 4, May 2019). See Section 7.1 [70] Describe the control group: In most studies, placebo VAC52150EBL3001 (Stage 2 only): MenACWY conjugate vaccine 7.6. List and provide frequency of Adverse Events of Special Interest Three cases of incident HIV infection in Ad26 participants in HIV-V-A004. For all three participants, factors known to increase the risk for HIV infection were present. All three events were assessed by the investigator as not related to study vaccination. HIV-1 infection is considered an AE of special interest in the Company’s HIV-1 clinical development program with the viral-vectored platform-based vaccines in (including Ad26-based vaccines), and a significant AE under monitoring for the other adenovirus vector-based programs. [32,70] (continued on next page) J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3097 7.7. Did Data Safety Monitoring Board (DSMB) or its equivalent oversee the study? Yes All studies are overseen by internal Data Review Committees (DRC) or external Independent Data Monitoring Committees (IDMC) or its equivalent. Did it identify any safety issue of concern? No IDMC’s/DRC’s did not identify issues. Potential issues were communicated to IDMC’s/DRC’s as per protocol requirements. If so describe: n.a. 8. Overall Risk Assessment of the Vector Information Comments/Concerns Reference[s] 8.1. Summarize key safety issues of concern identified to date, if any: No significant safety issues have been identified. How should they be addressed going forward: n.a. 8.2. What is the potential for causing serious unwanted effects and toxicities in: Describe the toxicities Please rate risk as: Healthy humans? Overall, the Ad26-based vaccines have been well tolerated, without significant safety issues identified. [70] Immunocompromised humans? The safety of Ad26.ZEBOV, an Ad26-based vaccine expressing the Ebola GP antigen, has been evaluated in HIV-positive participants on antiretroviral therapy (ART) with CD4+ counts of >350 and >200 cells/mL in two separate studies (VAC52150EBL2002 and VAC52150EBL2003 respectively). In study VAC52150EBL2002, 118 HIV-positive adult participants received a dose of Ad26.ZEBOV at 5 10 10 vp. In study VAC52150EBL2003, 221 adult participants received a dose of Ad26.ZEBOV at 5 10 10 vp. The vaccine was well tolerated in terms of local and systemic solicited and nonsolicited events, AEs with no SAEs or SUSARs in both studies. The safety and tolerability profile of the vaccine was similar between HIV-positive and HIVnegative participants. Low risk; HIV-infection was an exclusion criterion in most studies. Exposure HIV-infected adults: VAC52150EBL2002: 118 HIV-infected adults; CD4+ cells >350 cells/mL VAC52150EBL2003: 221 HIV-infected adults; CD4+ cells >200 cells/mL VAC89220HTX1001: 17 HIV-infected adults; CD4+ cells >400 cells/mL VAC89220HTX1002 (actively enrolling): 20 HIV-infected adults; CD4+ cells >350 cells/mL [70] Human neonates, infants, children? The AdVac safety database (version 4, May 2019) includes safety data from a total of 218 children between (4 to 17 years of age) who were vaccinated with Ad26.ZEBOV in VAC52150EBL2002. Overall, no safety concerns were identified in children after vaccination with Ad26.ZEBOV. Low risk; in seven clinical studies and vaccination campaigns, either ongoing or completed, with a last update of 4 September, >40,000 children (aged 0 to 17 years) were vaccinated with an Ad26based vaccine (cut-off: 4 September 2020; active only, estimate based on the study randomization ratios). So far, no safety concerns have been identified. Elderly The AdVac safety database (version 4, May 2019) includes safety data from a total of 180 elderly (60 years of age in stable health) who were vaccinated with Ad26.RSV.preF in VAC18193RSV2003. A total of 13 elderly (>64 years of age) participants were also enrolled in VAC52150EBL2002. Overall, no safety concerns were identified in elderly after vaccination with adenobased vaccines. Low risk; In three clinical studies either ongoing or completed with a last update of 21 December 2018, >32,500 elderly participants (60 years of age) were vaccinated with an Ad26-based vaccine (cut-off: 1 July 2020; active only, estimate based on the study randomization ratios). No safety concerns were identified. Pregnancy and in the unborn in humans? The most recent aggregate review of pregnancy exposure data was performed in September 2019; this analysis of the current experience with pregnancies after exposure to the Ebola candidate vaccines (Ad26.ZEBOV, Ad26.Filo) in female participants or partners of male participants did not reveal a safety concern. Serious complications or SAEs during pregnancy were reported in 20 out of a total of 66 pregnancies reported in female study participants. None of these serious complications/SAEs were considered causally associated with the study vaccines by Investigators or the Company. No apparent concernable pattern of AEs is emerging from this Low risk; pregnancy is an exclusion criterion for all Ad26-based vaccine studies except 1 study (described below). Pregnancy tests prior to vaccination and the use of adequate contraception was mandatory for all female participants of childbearing potential. Pregnant women are being enrolled in the ongoing Ebola vaccination study in DRC (DRC-EB-001/EBL3008). [71] J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3098 vaccine insert [4–8], which limits the use of Ad5-based vectors in human populations worldwide and encourages the development of recombinant adenoviral vectors based on other adenoviral serotypes. In contrast to Ad5-based vaccines, a similarly clear impact of naturalor vector-induced pre-existing immunity to Ad26 on vaccine immunogenicity has not been observed to date in clinical studies. Results from clinical trials assessing repeated administration of Ad26-based vaccination approaches showed that a second or subsequent dose of study vaccine was able to boost humoral and cellular immune responses to HIV antigens (Ad26.EnvA, study IPCAVD001; [28] and Ad26.Mos.HIV, APPROACH study [32]; even in the presence of high Ad26 neutralizing antibody titers induced by the first dose [28]. Similarly, naturally occurring Ad26 neutralizing antibody titers or Ad26-targeting T cell responses at baseline were not associated with decreased immune responses against the vaccine antigen [29,31]. Very extensive data and evidence have been collected on preclinical immunogenicity and efficacy, preclinical safety, clinical safety and immunogenicity as well as manufacturing and release, supporting the further evaluation and use of Ad26-based vectors for vaccine development. Recently, with a number of partners, the development of a COVID-19 vaccine has started in response to the pandemic caused by the SARS-CoV-2 virus. The prophylactic vaccine, which is based on the Ad26 vector expressing a variant of the SARS-CoV-2 spike protein, has entered in phase 3 clinical trial in September 2020; at the same time, manufacturing is being scaled up with the goal to supply large quantities of vaccine doses across the world [75]. 3. 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). 4. Purpose of document Risk benefit assessment of the Ad26 replication-incompetent viral vector using the Brighton Collaboration V3SWG Standardized Template for Collection of Key Information for Risk Assessment of Viral Vaccine Vector Candidates. Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The Brighton Collaboration V3SWG authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Jerome Custers, Maarten Leyssen, Frank Tomaka, Esther Heijnen, Georgi Shukarev, Dirk Heerwegh, Roy van Heesbeen, Hanneke Schuitemaker, and Macaya Douoguih, are current employees of Janssen Pharmaceuticals and potentially hold stock in J&J. Acknowledgements We thank the additional V3SWG members and participants for their support and helpful comments, and Patrick Zuber of the World Health Organization for bringing together many of the coauthors for completing the first draft of this template and for his support of the V3SWG. We would also like to thank Wouter Koudstaal (independent scientific writer) and Kerstin Luhn, Benoit Callendret, Maria Pau, Gert Scheper, Jerry Sadoff, Roland Zahn, Ad Knaapen, Jenny Hendriks, Myra Widjojoatmodjo, Marc Ceuppens, Raphaele Roten and Valerie Oriol Mathieu from Janssen for their role in compound development and manuscript preparation, Dan Barouch for his role in the development of Ad26-based vaccine candidates and the Brighton Collaboration Reference Group for their peer review. Funding for development of Ad26.ZEBOV for Ebola, Ad26.Mos. HIV, Ad26.Mos4.HIV, and Ad26.ENVA.01 for HIV, Ad26.CS.01 for Malaria, Ad26.RSV.FA2 and Ad26.RSV.preF for RSV, Ad26.Filo for Filovirus infection, Ad26.ZIKV.001 for Zika, Ad26.HPV16 and Ad26.HPV18 for HPV including generation of many data presented in the template, was provided by Janssen and/or partners, including in kind support. Main partners are Biological Advanced Research and Development Agency (BARDA), Innovative Medicine Initiative (IMI), Bill and Melinda Gates Foundation (BMGF), Military Health Research Program (MHRP), Beth Israel Deaconess Medical Center, Harvard Medical School (BIDMC), HIV Vaccine Trials Network (HVTN), Ragon Institute and the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIAID/NIH). Completion of this template was supported by WHO Registration Purchase Order Unit Reference 2019/936983-1 202338899-1MVP/EMPISAVISAV. Authorship All authors attest they meet the ICMJE criteria for authorship. References [1] Monath TP, Fast PE, Modjarrad K, Clarke DK, Martin BK, Fusco J, et al. rVSV D GZEBOV-GP (also designated V920) recombinant vesicular stomatitis virus pseudotyped with Ebola Zaire glycoprotein: standardized template with key considerations for a risk/benefit assessment. Vaccine X 2019;1:100009. review. No congenital malformations were reported to date in foetuses or newborns. Spontaneous abortion was the most commonly observed SAE (9 out of 66 pregnancies) with an incidence of 13.6%, which is within the range of expected spontaneous abortion rates during the first trimester of gestation, even when considering that spontaneous abortion incidences vary significantly depending on geographical areas and individual risk factors (e.g. age, previous abortions). In any other special populations n.a. 8.3. What is the potential for shedding and transmission in risk groups? There is no significant risk for shedding and transmission of Ad26-vectored vaccines across the risk groups (e.g. immunocompromised) who have received the vaccine vector. J. Custers, D. Kim, M. Leyssen et al. Vaccine 39 (2021) 3081–3101 3099 [2] Rosen L, Baron S, Bell JA. Four newly recognized adenoviruses. Proc Soc Exp Biol Med 1961;107:434–7. 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[7] Cheng C, Wang L, Gall JG, Nason M, Schwartz RM, McElrath MJ, et al. Decreased pre-existing Ad5 capsid and Ad35 neutralizing antibodies increase HIV-1 infection risk in the Step trial independent of vaccination. PLoS One 2012;7(4): e33969. [8] Catanzaro AT, Koup RA, Roederer M, Bailer RT, Enama ME, Moodie Z, et al. Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 candidate vaccine delivered by a replication-defective recombinant adenovirus vector. J Infect Dis 2006;194(12):1638–49. [9] Abbink P, Lemckert AA, Ewald BA, Lynch DM, Denholtz M, Smits S, et al. Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D. J Virol 2007;81(9):4654–63. [10] Mast TC, Kierstead L, Gupta SB, Nikas AA, Kallas EG, Novitsky V, et al. 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