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

doi:10.1016/j.vaccine.2020.09.018

PMC full text:

Vaccine. 2020 Oct 3

doi: 10.1016/j.vaccine.2020.09.018 [Epub ahead of print]

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 self-limiting. 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 non-integrating 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], [37], [38], [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 tract, in line with first isolation of Ad26 wild type from an anal specimen.	[40], [41]
• In immunocompromised humans	Limited information for Ad26 but human adenovirus infections has been documented in patients undergoing chemotherapy, allogenic stem cell transplantation or in patients with congenital or acquired immune deficiencies.	One case has been described of disseminated Ad26 infection in an immunocompromised individual with a severe brain tumour and irradiation history causing meningoencephalitis. Ad26 was associated with diarrhoea in a patient with acquired immune deficiency syndrome (AIDS).	[41], [42], [43]
• In human neonates, infants, children	Wild type Adenovirus type 26 has been isolated in 1956 from an anal specimen of a 9-month-old child. The child yielding Ad26 displayed minor illness; however, the illness could not be aetiologically associated with the isolated adenovirus.		[2]
• During pregnancy and in the unborn in humans	Unknown
• In any other special populations?	Unknown		[43]
3.8. What cell types are infected and what receptors are used in the natural host and in humans?	Ad26 can infect a wide range of cell types in vitro. Receptor use in humans (natural host) in vivo is not known.	The adenoviral life cycle (generally <2 days) starts with binding of the viral particle via the viral fibre knob to cell surface receptors; for most of the adenoviruses, this is the coxsackievirus B and adenovirus receptor (CAR). Ad26 is reported to use CD46 as the primary cellular receptor, but more recent reports indicated only a limited interaction between Ad26 and CD46, and even showed evidence of a role for αvβ3 integrins for efficient transduction of epithelial cells or interactions with sialic acids.	[9], [18], [19], [20]
3.9. What is known about the mechanisms of immunity to the wild type virus?	Little is known about the mechanisms of immunity to wild-type adenovirus. However, neutralizing antibodies can inhibit viral entry in vitro. In addition, in immunocompromised people having invasive adenovirus infections, the adoptive transfer of adenovirus-specific T-cells has been successful, suggesting an important role for T-cells in immunity to human adenovirus.		[42], [44]
3.10. Has disease enhancement been demonstrated with the wild type virus:
• In vitro?	No
• In animal models?	No
• In human hosts?	No
3.11. Is antibody-dependent enhancement (ADE) a possible contributor to the pathogenesis of wild type disease?	Not reported
3.12. What is the background prevalence of natural immunity to the virus?	In humans, depending on the geographical location, 10–90% of the individuals tested have neutralizing antibodies against Ad26. However, neutralization titres are low-intermediate compared to what has been observed for other human adenovirus types, as with Ad5.		[9], [10], [11], [12], [13]
3.13. Is there any vaccine available for the wild-type virus? If yes:	No
• What populations are immunized?	n.a.
• What is the background prevalence of artificial immunity?	n.a.
3.14. Is there treatment available for the disease caused by the wild type virus?	No specific treatments for human adenovirus infections are available.	Ribavirin and cidovir have been used to treat immunocompromised individuals. For Ad4, cidofovir and brincidofovir are effective in vitro and have appeared effective in clinical trials.	[45], [46]
4. Characteristics of the vector from which vaccine(s) may be derived	Information	Comments/Concerns	Reference[s]
4.1. Describe the source of the vector (e.g. isolation, synthesis)	The Ad26 vector is based on human adenovirus type 26, group D. Adenovirus type 26 wild-type virus has been isolated in 1956 from an anal specimen of a 9-month-old child. To create the final Ad26 vector, two cloning plasmids were used, one containing the left arm of the genome, and the second containing the right arm of the genome. These plasmids were used to introduce modifications to the Ad26 vector genome. These modifications include deletion of the complete E1a and E1b region, partial deletion of the E3 region, which is non-essential for in-vitro replication. In addition, Ad26 E4 open reading frame (orf) 6 was exchanged for those of Ad5 to allow production of replication incompetent Ad26 vectors on Ad5 E1 complementing cell lines, i.e., PER.C6® cells. A transgene expression cassette is placed in the E1-deleted region containing a human cytomegalovirus (CMV) immediate-early promoter and a simian virus 40 (SV40) derived polyadenylation (pA) signal for strong expression of a foreign transgene.		[2], [9]
4.2. What is the basis of attenuation/inactivation of the wild type virus to create the vector?	Ad26 was rendered replication incompetent by deletion of early region 1 (ΔE1). A partial deletion of non-essential early region 3 (ΔE3) was made to create enough space in the genome for the transgene expression cassette (inserted in E1 region) also further attenuating the vector.		[9]
4.3. What is known about the replication, transmission and pathogenicity of the vector in humans in the following categories?:	The Ad26 vector is replication incompetent in non-E1 complementing human cells.
• In healthy people	n.a.
• In immunocompromised people	n.a.
• In neonates, infants, children	n.a.
• During pregnancy and in the unborn	n.a.
• In gene therapy experiments	n.a.
• In any other special populations	n.a.
4.4. Is the vector replication-competent in non-human species?	The Ad26 vector is replication incompetent in non-E1 complementing cells and, as such, will be replication incompetent when administered to non-human species. In addition, insertion of foreign antigens is not expected to change the host-range of the vaccine vector.
4.5. What is the risk of reversion to virulence or recombination with wild type virus or other agents?	Recombination of Ad26 vaccine vectors with wild-type viruses would require sequence homology and presence of both the genome of Ad26 vaccine vectors and wild-type adenoviruses to be present in the same cell(s). Nonclinical biodistribution studies show that adenoviral vector DNA of Ad26 vaccine vectors did not distribute widely, as the vector DNA was primarily detected at the site of administration in the muscle, the draining lymph nodes and, to a lesser extent, to the spleen. DNA of intramuscularly administered replication-incompetent Ad vaccine vector and wild-type adenovirus DNA are unlikely to co-locate in the same body compartments. In the unlikely event that recombination occurs between vaccine vector and wild-type adenoviruses, the virulence can maximally be equal to the wild-type adenovirus already present in the tissue. The majority of the theoretical homologous recombination products are replication incompetent or attenuated forms of the Ad26. Reversion of Ad26 virulence by recombination is therefore highly unlikely. Furthermore, reversion of virulence due to nucleotide mutations is impossible since deletion of the E1 gene from the Ad26 vector cannot be restored by random mutations and or indels. Recombination with other viruses has not been described and is considered highly unlikely due to the limited biodistribution and absence of sequence homology and replication.
4.6. Is the vector genetically stable in vitro and/or in vivo?	The dsDNA genome of Ad26 virus is relatively stable when compared to RNA viruses. Genetic stability of the vector is confirmed during manufacturing and upscaling by extended passaging and/or genetic stability testing.		[47]
4.7. What is the potential for shedding and transmission to humans or other species?	Vector shedding is limited and transmission of the Ad26 vector is highly unlikely in view of: (i) the vector is replication-incompetent, and thus, allows only for one-time transduction of the target cell, (ii) the limited shedding as observed in clinical studies, (iii) the limited biodistribution profile as observed in nonclinical studies, and (iv) the very low probability of regaining replication-competence through recombination with co-infecting wild-type virus.
4.8. Does the vector establish a latent or persistent infection?	Biodistribution studies in rabbits have shown that vector DNA is not widely distributed, and clearance has been observed indicating that the vector is unlikely to persist in the tissues following intramuscular injection.	In nature, wild-type adenovirus is known to be able to cause persistent infections. Whether the Ad26 vector can persist for longer time in humans is unknown.
4.9. Does the vector replicate in the nucleus?	The vector is replication incompetent.
4.10. What is the risk of integration into the human genome?	See 3.6.		[36], [37], [38], [39]
4.11. Is there any previous human experience with this or a similar vector (safety and immunogenicity records)?	Yes, numerous vaccine clinical studies have been performed with Ad26-based replication incompetent vectors (see section 7).		[27], [28], [29], [30], [31], [32], [48], [49], [50]
4.12. What cell types are infected and what receptors are used in humans?	See 3.8. The Ad26 vector is expected to have the same cell tropism as the wild-type Ad26 virus. Receptor use in humans in vivo is unknown.		[9], [18], [19], [20]
4.13. What is known about the mechanisms of immunity to the vector?	Little is known about the mechanisms of immunity to the vector. However, neutralizing antibodies and cellular responses are induced after Ad26 vector administration to humans and non-human species. Vector specific neutralizing antibodies can specifically inhibit vector entry in vitro. In vivo, little to no indication that neutralizing antibodies inhibit the vector has been found.		[42], [44]
4.14. Has ADE been demonstrated with the vector:
• In vitro?	No
• In animal models?	No
• In human hosts?	No
4.15. Is there antiviral treatment available for disease manifestations caused by the vector?	The vector is replication-incompetent; thus, no disease manifestations as seen with wild-type infection are expected. Therefore, there is no need for antiviral treatment.
4.16. Can the vector accommodate multigenic inserts or will several vectors be required for multigenic vaccines?	Ad26 can accommodate multigenic inserts, theoretical maximum total insert size up to 8,6 kb (approx. 105% of wild-type Ad26 genome size). In specific cases, multiple vectors may be required to accommodate multigenic vaccines.	As an example, the 4-valent prophylactic HIV vaccine Ad26 component consists of 4 ‘monogenic’ Ad26 vectors combined in one vaccine vial.	[51]
5. Characteristics of vector-based vaccine(s)	Information	Comments/Concerns	Reference[s]
5.1. What is the target pathogen?	Ad26 can serve as a vaccine vector for any target pathogen.
5.2. What is identity and source of the transgene?	Antigens of HIV-1 (Env, gag, pol), RSV (FA2, preF), Ebola virus (GP), Sudan virus (GP), Marburg virus (GP), Zika virus (M−E), Human papilloma virus (E2, E6, E7), Plasmodium falciparum (CSP) have been inserted in the replication incompetent Ad26 vector and, have been or, are being tested and evaluated in humans in several clinical trials.		[27], [28], [29], [30], [31], [32], [48], [49], [50], [74], [52], [54], [55], [56], [57], [58], [59], [60], [61]
5.3. Is the transgene likely to induce immunity to all strains/genotypes of the target pathogen?	This will depend on the design of the antigen and the antigenic diversity of the pathogen.
5.4. Where in the vector genome is the transgene inserted?	The transgene is inserted in the E1 region at the site of the E1-deletion.		[9]
5.5. Does the insertion of the transgene involve deletion or other rearrangement of any vector genome sequences?	The E1 region is deleted to render the vector replication incompetent and together with a deletion in the E3 region provide space for insertion of a transgene expression cassette.		[9]
5.6. How is the transgene expression controlled (transcriptional promoters, etc.)?	In general, expression of the antigen is regulated using the long human CMV immediate-early promoter, which is thought to be active in most mammalian cells, and an SV40-derived polyadenylation sequence.		[62]
5.7. Does insertion or expression of the transgene affect the pathogenicity or phenotype of the vector?	The expressed antigen is not part of the viral particle and, as such, it is not expected that the phenotype of the vector nor the pathogenicity (the vector is replication incompetent) of the vector are altered.
5.8. Is the vaccine replication-competent in humans or other species?	The Ad26 vector is replication incompetent.
5.9. What is the risk of reversion to virulence or recombination with wild type or other agents?	See 4.5.
5.10. Is the vaccine genetically stable in vitro and/or in vivo?	Genetic stability of the vaccine vector is confirmed during manufacturing and upscaling by extended passaging and genetic stability testing.
5.11. What is the potential for shedding and transmission to humans or other species?	See 4.7.
5.12. Does the vaccine establish a latent or persistent infection?	The vaccine is replication incompetent and is unable to establish a productive infection. Persistence/latency is not expected as the vaccine misses the E1 and E3 genes that code for proteins involved in countering the host immune system. In addition, nonclinical biodistribution studies of the vaccine have shown that the Ad26 vaccine vector is cleared from vector positive tissues (see 6.7.).
5.13. Does the vaccine replicate in the nucleus?	The vaccine is replication incompetent. The vector genome (linear ds-DNA) travels to the nucleus of the host cell where antigen expression occurs, in the absence of vaccine vector replication.
5.14. What is the risk of integration into the human genome?	See 3.6.		[36], [37], [38], [39]
5.15. List any disease manifestations caused by the vaccine in humans, the strength of evidence, severity, and duration of disease for the following categories:	n.a.
• In healthy people	n.a.
• In immunocompromised people	n.a.
• In neonates, infants, children	n.a.
• During pregnancy and in the unborn	n.a.
• In any other special populations	n.a.
5.16. What cell types are infected and what receptors are used in humans?	See 3.8. The Ad26 vector is expected to have the same cell tropism as the wild-type Ad26 virus.		[9], [18], [19], [20]
5.17. What is known about the mechanisms of immunity to the vaccine?	In general, the immune responses to the vaccine antigen encoded by the vector are characterized by a rapid increase in binding and in most cases neutralizing antibodies. In addition, induction of non-neutralizing, functional antibodies with effector functions, like antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), have been observed. Induction of cellular immunity (both CD4+ and CD8+ T-cells) is also observed.		[27], [28], [29], [30], [31], [32], [74], [52], [54], [55], [56], [57], [58], [59], [60], [61]
5.18. Has disease enhancement been demonstrated with the vaccine:
• In vitro?	No
• In animal models?	No		[55]
• In human hosts?	No
5.19. What is known about the effect of pre-existing immunity, including both natural immunity and repeat administration of the vector or the vaccine, on ‘take’, safety or efficacy in any animal model or human studies using this vector?	Data acquired to date, in more than 6,000 vaccinated human participants, have not revealed impact of pre-existing vector immunity on the vaccine insert specific humoral or cellular response. Repeated administration with the Ad26 vector leads to an increase in antigen specific humoral responses and a maintenance of cellular responses. With more than 114,000 participants vaccinated overall, no safety issues have been identified.		[28], [32]
5.20. Is the vaccine transmissible in humans or other species (including arthropods) and/or stable in the environment?	The vaccine is replication incompetent, so no vaccine transmission is expected (see also 4.7.).
5.21. Are there antiviral or other treatments available for disease manifestations caused by the vaccine?	The vector is replication-incompetent; thus, no disease manifestations are expected besides local and systemic reactogenicity. Therefore, there is no benefit for antiviral treatment.
5.22. Vaccine formulation	Liquid formulation.
5.23. Proposed route of vaccine administration	Intramuscular administration.
5.24. Target populations for the vaccine (e.g. paediatric, maternal, adult, elderly etc.)	Most target populations can be envisioned.
6. Toxicology and potency (Pharmacology) of the vector	Information	Comments/Concerns	Reference[s]
6.1. What is known about the replication, transmission and pathogenicity of the vector in and between animals?	The Ad26 vector does not replicate and does not cause clinical illness in animals.	The Ad26 vector showed a limited distribution following intramuscular injection in rabbits and clearance of the vector was observed, indicating that the vector does not replicate and/or persist in the tissues following intramuscular (IM) injection. The Ad26 vector did not induce any adverse effects in multiple GLP repeat-dose toxicity studies in rabbits (and rats), irrespective of the transgene insert used.
6.2. For replicating vectors, has a comparative virulence and viral kinetic study been conducted in permissive and susceptible species? (yes/no) If not, what species would be used for such a study? Is it feasible to conduct such a study?	n.a., since the Ad26 vector is replication incompetent.
6.3. Does an animal model relevant to assess attenuation exist?	n.a., since the Ad26 vector is replication incompetent.
6.4. Does an animal model for safety including immuno-compromised animals exist?	n.a., since the Ad26 vector is replication incompetent.
6.5. Does an animal model for reproductive toxicity exist?	n.a., since the Ad26 vector is replication incompetent.	The general (repeat-dose) toxicity studies conducted with the replication incompetent Ad26 vector in rabbits (and rats) have not revealed any effects on male sex organs that would impair male fertility. In addition, the general (repeat-dose) and/or developmental and reproductive toxicity studies did not reveal any evidence of impaired female fertility nor did not indicate harmful effects with respect to reproductive toxicity in female rabbits.
6.6. Does an animal model for immunogenicity and efficacy exist?	Most mammalian species studied to date have shown induction of insert specific immunity after administration of Ad26-based vaccines, dependent on the studied disease. Several efficacy animal models exist – the most studied animal models have been mice, rabbits, ferrets, cotton rats and several non-human primate (NHP) species.
6.7. Does an animal model for antibody enhanced disease or immune complex disease exist?	No
6.8. What is known about biodistribution in animal models or in humans?	Biodistribution studies have been conducted in rabbits. Following intramuscular administration, the Ad26 vector did not widely distribute as vector DNA was primarily detected at the site of injection, draining lymph nodes and (to a lesser extent) the spleen. Over time, the number of animals with positive tissues and/or the vector copy number present in those positive tissues declined, indicating clearance of the Ad26 vector.
6.9. What is the evidence that vector derived vaccines will generate a beneficial immune response in:
• Small animal models?	Mice, cotton rats, rabbits and ferrets have been shown to develop immune responses against a variety of vaccine inserts.		[9], [52], [54], [55], [58], [63], [64], [65], [66], [67]
• Nonhuman primates (NHP)?	NHP have been shown to develop protective immune responses against a variety of vaccine inserts.		[9], [18], [53], [57], [58], [60], [61], [65], [68], [69]
• Human?	Several vaccine inserts in this vector have been shown to be immunogenic in a broad range of populations based on age and location.		[27], [28], [29], [30], [31], [32], [48], [49], [50], [51]
6.10. Have challenge or efficacy studies been conducted in subjects with:
• Healthy humans?	Efficacy studies Efficacy studies are ongoing for Janssen’s HIV and RSV vaccine candidates: HIV VAC89220HPX2008 - HIV-1-uninfected women aged 18 to 35 years VAC89220HPX3002 - HIV-1-uninfected cis-gender men and transgender individuals having sex with cis-gender men and/or transgender individuals, aged ≥18 to ≤60 years RSV VAC18193RSV2001 - Older adults ≥65 years Challenge studies: Challenge studies have been performed with in malaria and RSV in healthy volunteers.RSV VAC18193RSV2002 - Healthy adults aged ≥18 to ≤50 years Malaria MAL-V-A001 - Healthy adults aged ≥18 to ≤50 years
• HIV positive	Studies in HIV positive participants: There have been no challenge or efficacy studies in HIV-positive participants. A therapeutic vaccination study is ongoing in HIV-positive participants in Thailand.	The immunogenicity of the Ad26.ZEBOV vaccine has been evaluated in HIV-positive and negative participants in two separate studies. The safety and tolerability profile of the vaccine was similar in HIV-positive and HIV-negative participants (see 8.2).
• Other diseases?	n.a.
6.11. Have studies been done simultaneously or sequentially administering more than one vector with different transgenes? Is there evidence for interaction/interference?	The prophylactic HIV vaccine is a combination of four vectors, where we have shown that the addition of the fourth vector over the trivalent combination enhanced responses. In a multivalent filovirus vaccine, responses to one out of three of the vaccines was unchanged compared to the single administration. Sequential administration has been shown to be immunogenic upon each following administration, with up to four administrations of the vector tested.		[29], [32], [60]
*7. Adverse Event (AE) Assessment of the Vector (see Instructions):**	Information	Comments/Concerns	Reference[s]
7.1. Approximately how many humans have received this viral vector vaccine to date? If variants of the vector, please list separately.	In total, more than 114,000 participants across 49 clinical studies and Rwanda government led vaccination campaigns have been vaccinated with an Ad26-based vaccine (cut-off: 4 September 2020). The AdVac safety database (version 4, May 2019) contains data from 23 clinical studies using Ad26-based vaccines. Safety data of a total of 3,694 adult participants and 218 children is included in the report of the database. The cut-off date of this report was 21 December 2018. Ebola program Ad26.ZEBOV: 2,668 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).		[70]
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 post-vaccination, regardless of time to onset.
• Solicited AEs	Yes	In most studies, solicited AEs were generally collected for a 7-day post-vaccination period, using a study participant diary.
• Unsolicited AEs	Yes	Unsolicited AEs were generally collected up to 28–30 days/4 weeks post-vaccination 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.
• 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). Injection-site 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]
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/µL in two separate studies (VAC52150EBL2002 and VAC52150EBL2003 respectively). In study VAC52150EBL2002, 118 HIV-positive adult participants received a dose of Ad26.ZEBOV at 5 × 10¹⁰ vp. In study VAC52150EBL2003, 221 adult participants received a dose of Ad26.ZEBOV at 5 × 10¹⁰ vp. The vaccine was well tolerated in terms of local and systemic solicited and non-solicited events, AEs with no SAEs or SUSARs in both studies. The safety and tolerability profile of the vaccine was similar between HIV-positive and HIV-negative 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/µL VAC52150EBL2003: • 221 HIV-infected adults; CD4+ cells >200 cells/µL VAC89220HTX1001: • 17 HIV-infected adults; CD4+ cells >400 cells/µL VAC89220HTX1002 (actively enrolling): • 20 HIV-infected adults; CD4+ cells >350 cells/µL	[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 Ad26-based 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 adeno-based 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 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).	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]
• 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.