Safety evaluation of the single-dose Ad26.COV2.S vaccine among healthcare workers in the Sisonke study in South Africa: A phase 3b implementation trial.

BACKGROUND: Real-world evaluation of the safety profile of vaccines after licensure is crucial to accurately characterise safety beyond clinical trials, support continued use, and thereby improve public confidence. The Sisonke study aimed to assess the safety and effectiveness of the Janssen Ad26.COV2.S vaccine among healthcare workers (HCWs) in South Africa. Here, we present the safety data. METHODS AND
FINDINGS: In this open-label phase 3b implementation study among all eligible HCWs in South Africa registered in the national Electronic Vaccination Data System (EVDS), we monitored adverse events (AEs) at vaccination sites through self-reporting triggered by text messages after vaccination, healthcare provider reports, and active case finding. The frequency and incidence rate of non-serious and serious AEs were evaluated from the day of first vaccination (17 February 2021) until 28 days after the final vaccination in the study (15 June 2021). COVID-19 breakthrough infections, hospitalisations, and deaths were ascertained via linkage of the electronic vaccination register with existing national databases. Among 477,234 participants, 10,279 AEs were reported, of which 138 (1.3%) were serious AEs (SAEs) or AEs of special interest. Women reported more AEs than men (2.3% versus 1.6%). AE reports decreased with increasing age (3.2% for age 18-30 years, 2.1% for age 31-45 years, 1.8% for age 46-55 years, and 1.5% for age > 55 years). Participants with previous COVID-19 infection reported slightly more AEs (2.6% versus 2.1%). The most common reactogenicity events were headache (n = 4,923) and body aches (n = 4,483), followed by injection site pain (n = 2,767) and fever (n = 2,731), and most occurred within 48 hours of vaccination. Two cases of thrombosis with thrombocytopenia syndrome and 4 cases of Guillain-Barré Syndrome were reported post-vaccination. Most SAEs and AEs of special interest (n = 138) occurred at lower than the expected population rates. Vascular (n = 37; 39.1/100,000 person-years) and nervous system disorders (n = 31; 31.7/100,000 person-years), immune system disorders (n = 24; 24.3/100,000 person-years), and infections and infestations (n = 19; 20.1/100,000 person-years) were the most common reported SAE categories. A limitation of the study was the single-arm design, with limited routinely collected morbidity comparator data in the study setting.
CONCLUSIONS: We observed similar patterns of AEs as in phase 3 trials. AEs were mostly expected reactogenicity signs and symptoms. Furthermore, most SAEs occurred below expected rates. The single-dose Ad26.COV2.S vaccine demonstrated an acceptable safety profile, supporting the continued use of this vaccine in this setting. TRIAL REGISTRATION: ClinicalTrials.gov NCT04838795; Pan African Clinical Trials Registry PACTR202102855526180.

Introduction

South Africa is among the countries most affected globally by COVID-19, with over 266,000 excess natural deaths occurring between May 2020 and October 2021 (approximately 448 per 100,000 individuals) [1]. The single-dose Ad26.COV2.S vaccine showed efficacy in preventing symptomatic and severe COVID-19 disease in the ENSEMBLE study including in South Africa, where initially the beta variant and then the delta variant were the predominant circulating strains [2,3]. Here, an estimated 1,300 healthcare workers (HCWs) have died from COVID-19 as of September 2021 [4]. The Sisonke study, an open-label, single arm phase 3b implementation study of the single-dose Ad26.COV2.S vaccine, was conducted as an emergency intervention to protect HCWs in the face of an anticipated third COVID-19 wave, at a time when no vaccines were available through the national rollout. The study aimed to assess the safety and effectiveness of the Janssen Ad26.COV2.S vaccine among HCWs in South Africa.

The Ad26.COV2.S vaccine is compatible with standard vaccine storage and distribution channels and is therefore a practical vaccine for low- and middle-income countries or remote populations [5]. To date, approximately 30 million persons in the United States (US) and the European Union have received the Ad26.COV2.S vaccine [6]. Vaccine adverse event (AE) surveillance systems demonstrate that billions of people have safely received COVID-19 vaccines [7]. AEs following COVID-19 vaccination are generally mild, and include local reactions, such as injection site pain, redness, swelling, and systemic reactions, like fever, headache, fatigue, nausea, vomiting, and diarrhoea [8,9].

As reported by the US Centers for Disease Control and Prevention, severe or potentially life-threatening AEs are rare, and after 12.6 million doses of the Ad26.COV2.S vaccine, 38 cases of thrombosis with thrombocytopenia syndrome (TTS) and 98 cases of Guillain-Barré syndrome (GBS) were reported, while after 141 million second mRNA vaccine doses, 497 cases of myocarditis were reported [10]. Following the precautionary pause instituted by the Food and Drug Administration (FDA) in April 2021, the South African Health Products Regulatory Authority recommended a similar 2-week pause for the Sisonke study [11]. The study recommenced with additional safeguards including screening and monitoring of participants at high risk of thrombosis and implementing measures to safely manage participants with TTS. Participant information sheets and informed consent forms were updated to include the newly identified AEs. Identification of such rare events illustrated that continued evaluation of the safety profile of vaccines post-licensure is crucial to accurately characterise safety and to identify very rare AEs that may not be reported in clinical trials.

The Sisonke study enrolled almost half a million HCWs, providing an opportunity to further evaluate the safety of the Ad26.COV2.S vaccine in an expanded population. Here we present the safety data.

Methods

Study participants

The Sisonke study is a multi-centre, open-label, single-arm phase 3b implementation study among HCWs (≥18 years) in South Africa, which is conducted in collaboration with the National Department of Health (ClinicalTrials.gov NCT04838795; Pan African Clinical Trials Registry PACTR202102855526180). All 1,250,000 HCWs targeted by phase 1 of the national COVID-19 Vaccine Rollout Strategy were invited for vaccination. To participate, HCWs were required to register on the national Electronic Vaccination Data System (EVDS) and provide electronic consent if they were age 18 or older. All eligible HCWs who registered on the EVDS and provided electronic consent for the study were eligible for enrolment. Known pregnant women and breastfeeding women were excluded due to a lack of sufficient safety data at that time. Details of the eligibility criteria are provided in the study protocol (S1 Appendix). A total of 477,234 HCWs received the Ad26.COV2.S vaccine between 17 February 2021 and 17 May 2021.

The institutional health research ethics committees of participating clinical research sites approved the study, which was overseen by the South African Health Products Regulatory Authority (Ref: 20200465). This study is reported as per the Consolidated Standards of Reporting Trials (CONSORT) guideline (S6 Appendix).

Vaccination procedures

Participants received appointments for vaccination through the EVDS or were invited via employer lists. Vaccinations were conducted in collaboration with the National Department of Health public or private vaccination centres across all 9 South African provinces and overseen by Good Clinical Practice–trained personnel linked to one of the ENSEMBLE trial research sites. Participants received a single intramuscular injection of Ad26.COV2.S at a dose of 5 × 1010 virus particles and were observed for AEs for 15 minutes post-vaccination, or for 30 minutes if they had a previous history of allergic reactions to vaccinations.

AE reporting

AEs were reported into the study database via multiple streams using a hybrid surveillance system that combined passive with active reporting [12]. First, we designed an electronic case report form (CRF) (S2 Appendix). After vaccination, every participant received a text message with COVID-19 infection prevention measures that also listed common signs and symptoms of reactogenicity and provided an AE reporting web link, which allowed participants access to the form for AE reporting. Reactogenicity events are pre-specified common AEs expected soon after vaccination and include systemic events such as headache, fever, myalgia, arthralgia, malaise, nausea, and chills, and local events such as pain, erythema, and induration. Second, healthcare providers were able to complete paper-based CRFs that were available at healthcare and vaccination facilities, which were then submitted to the Sisonke study safety desk and captured in the AE database. Third, the study team set up a safety desk call centre staffed by pharmacovigilance nurses, pharmacists, and safety physicians to assess and advise on AE reports. Contact details were advertised on the vaccination cards, shared on social media, and included in the text messages. Finally, spontaneous case reports via unsolicited communication by HCWs were captured and verified by safety desk staff. Telephone follow-up with the participant and attending healthcare provider was established as part of case investigation. Case reports for safety events of concern were collated from these telephone interviews, medical records, and results from laboratory and imaging investigations.

In addition, we actively linked EVDS data via national identification numbers with national patient-level disease databases, COVID-19 case notifications, and the national population registry to identify vaccine recipients with COVID-19 infections, COVID-19-related hospitalisations, and deaths. COVID-19 is a notifiable medical condition in South Africa, and tests conducted across laboratories are reported to the National Health Laboratory Service data system, which was used to identify seropositive Sisonke study participants via active linkage. A death notification form must be submitted to the Department of Home Affairs to obtain a death certificate. Therefore, in addition to case reports and active tracing, mortality was ascertained via linkage with the national population registry. After identification of deaths, the safety staff contacted next of kin and primary healthcare providers and solicited medical records to ascertain cause of death.

Safety monitoring

AE reports were processed daily and screened for serious AEs (SAEs); SAEs were defined by the investigators as any AE that results in death, is life-threatening, requires inpatient hospitalisation or prolongation of existing hospitalisation, results in persistent or significant disability/incapacity, or is a congenital anomaly/birth defect. AEs of special interest (AESIs) were defined per the Brighton Collaboration list [9] (hereafter SAEs and AESIs are referred to collectively as SAEs). Anaphylaxis was adjudicated using the Brighton Collaboration and National Institute of Allergy and Infectious Diseases case definition, with cases needing to meet both definitions to be considered confirmed cases [13,14]. Seven days after reporting an AE, participants received a follow-up text message with a link to the electronic CRF. Participants reporting worsening or non-resolving symptoms were followed up by safety desk staff. After the FDA lifted the cautionary 2-week pause in vaccinations, 2 additional follow-up text messages were sent to all participants 7 and 14 days after vaccination. The texts highlighted signs/symptoms associated with TTS and provided a link to the electronic CRF. Safety staff made attempts to obtain medical records and supporting information from healthcare providers for all reported SAEs.

The protocol safety review team comprising principal investigators, safety physicians, and subject matter experts (haematologists, neurologist, allergy expert, and infectious disease specialists) provided oversight by weekly safety data review. An independent safety monitoring committee provided additional safety oversight.

Statistical analysis

A prospective analysis plan was used in designing the study (see S3 Appendix). For descriptive statistics, counts and proportions were used for categorical variables, and medians and interquartile ranges for quantitative variables. The baseline characteristics sex, age, previous COVID-19 status, and presence of comorbidities were compared between participants reporting AEs and those not reporting AEs using descriptive statistics. SAEs were summarised by MedDRA system organ class and adverse event preferred term. For selected SAEs, disproportionality analysis was conducted: The observed (O) number of reported cases was compared to the expected (E) number based on background incidence rates, and the O/E ratio with 95% confidence interval was calculated. The 95% confidence intervals for the O/E ratio were calculated as follows: (i) the 95% confidence interval of the observed number of events was calculated assuming a chi-squared distribution, and (ii) each of the lower and upper limits of the confidence interval in (i) were divided by the number of expected events to produce the 95% confidence interval for the O/E ratio. Available background incidence rates were used including a medically insured population in South Africa (see S4 Appendix) for pulmonary embolism and deep venous thrombosis, a Tanzanian population-based cohort study for neurological events such as stroke, the Brighton Collaboration, and European population databases [10,15-18]. Person-time was accrued from the date of vaccination until death or dataset closure on 15 June 2021 (28 days after the last vaccination in the study). The incidence rates per 100,000 person-years were calculated using a Poisson model with person-years as an offset. Comparison of age-standardised mortality rates in the Sisonke study was with projected background population mortality rates in South Africa as per the 2018 Medical Research Council Rapid Mortality Surveillance Report [19] and pre-COVID-19 local employee group life assurance data for a similar age-structured working population. Deaths were excluded from the SAE analysis and examined as a separate entity. COVID-19-related deaths are published in a separate effectiveness report [20]. There were no formal statistical methods that were used to handle missing data. All statistical analyses were conducted using Stata version 14 (StataCorp, College Station, TX, US). See S5 Appendix for the analytical code.

Results

Participants

The Sisonke study enrolled and vaccinated 477,234 participants from all 9 South African provinces between 17 February 2021 and 17 May 2021 (all 1,250,000 HCWs in the country were invited to participate). The majority were women (74.9%), the median age was 42 years (IQR 33–51), and 16.3% were older than 55 years. Previous COVID-19 infection was self-reported by 14.5% of participants. The most prevalent comorbidities were hypertension (15.6%), HIV infection (8.3%), and diabetes mellitus (5.9%) (Table 1).

Table 1

Demographic and clinical characteristics of Ad26.COV2.S vaccine recipients in the Sisonke study.

Characteristic	Total number* (n = 477,234)	Number* reporting AEs	Percent (95% CI)* reporting AEs	p-Value
Sex				<0.001
Female	357,481	8,389	2.3 (2.30–2.40)
Male	119,753	1,890	1.6 (1.51–1.65)
Age (median, IQR)	42	38	30–48	<0.001
Age category (years) **				<0.001
18–30	85,486	2,697	3.2 (3.04–3.27)
31–45	209,376	4,484	2.1 (2.08–2.20)
46–55	104,078	1,902	1.8 (1.75–1.91)
>55	78,162	1,195	1.5 (1.45–1.62)
Previous COVID-19				<0.001
No	408,085	8,478	2.1 (2.03–2.12)
Yes	69,149	1,801	2.6 (2.49–2.73)
Comorbidities
Hypertension				<0.001
No	402,853	8,846	2.2 (2.15–2.24)
Yes	74,381	1,433	1.9 (1.83–2.03)
Diabetes				0.003
No	449,171	9,744	2.2 (2.13–2.21)
Yes	28,063	535	1.9 (1.75–2.07)
HIV				<0.001
No	437,848	9,800	2.2 (2.19–2.28)
Yes	39,386	479	1.2 (1.11–1.33)
Cancer				0.528
No	475,870	10,253	2.2 (2.11–2.20)
Yes	1,364	26	1.9 (1.30–2.78)
Previous tuberculosis				0.047
No	476,756	10,275	2.2 (2.11–2.20)
Yes	478	4	0.8 (0.31–2.21)
Heart disease				0.232
No	473,804	10,195	2.2 (2.11–2.19)
Yes	3,430	84	2.4 (1.98–3.02)
Chronic lung disease				<0.001
No	475,501	10,197	2.1 (2.10–2.19)
Yes	1,733	82	4.7 (3.83–5.84)

AE, adverse event; IQR, interquartile range; 95% CI, 95% confidence interval; HIV, human immunodeficiency virus.

*Except for age, for which median or IQR is given.

**Missing values: age category, n = 132.

AEs reported by baseline characteristics

A total of 10,279 AEs were reported, of which 138 (1.4%) were classified as SAEs. Most AE reports (81%) were electronic self-reports. Women reported more AEs than men (2.3% versus 1.6%; p < 0.001), AE reporting decreased with increasing age (3.2% for 18- to 30-year-olds versus 1.5% for >55-year-olds; p < 0.001), and participants with previous COVID-19 infection reported more AEs (2.6% versus 2.1%; p < 0.001). Persons with HIV (1.2% versus 2.2%; p < 0.001) or previous tuberculosis (0.8% versus 2.2%; p = 0.043) reported fewer AEs than those without, while more AEs were reported by participants with chronic lung disease compared to those without (4.7% versus 2.1%; p < 0.001). Proportions reporting AEs among those with other comorbidities were similar (Table 1).

Most reported AEs (n = 9,021, 81%) were reactogenicity events; the most common were headaches and body aches (including arthralgia, myalgia, and fatigue) that occurred within the first 7 days of vaccination, followed by mild injection site pain and fever (Fig 1). These events occurred predominately on the day of vaccination, reducing in frequency by day 2. A total of 2,109 AEs were not classified as reactogenicity events. Self-reported severity was mild to moderate in 67% of participants (n = 7,157), i.e., the event did not result in loss of ability to perform usual social and functional activities, while 32% of participants (n = 3,375) reported being unable to perform usual activities and 2% (n = 213) reported that they needed to visit the emergency room or were hospitalised. Follow-up at day 7 post-vaccination indicated that of the 3,831 who had responded by dataset closure, 92% (3,524) of participants reporting AEs had either completely recovered or were recovering. The remaining 8% (324) of participants were contacted by the safety team. Attempts were made by the safety team to contact all of these participants individually (3 telephone calls at least 1 day apart). Those who were contactable were interviewed via phone and, if clinically indicated, referred for further care.

Fig 1

Commonly occurring adverse events in the first 7 days post-vaccination.

Serious adverse events

A total of 138 SAEs (excluding deaths) were reported by 136 participants (median age 42 years, IQR 35–51), with 113 (81.9%) reported by women and 25 (18.1%) by men. Most SAEs (115; 82.7%) occurred within 28 days of vaccination, with a median time to onset of 5 days (IQR 1–17) for all SAEs, and a median time of onset of 1 day (IQR 0–9) for SAEs occurring with 28 days of vaccination. Vascular (n = 37; 39.1/100,000 person-years) and nervous system disorders (n = 31; 31.7/100,000 person-years), immune system disorders (n = 24; 24.3/100,000 person-years), and infections and infestations (n = 19; 20.1/100,000 person-years) were the most common reported SAE categories (Table 2). SAE outcomes were as follows: 48 (34.8%) recovered, 36 (26.1%) recovering, and 9 (6.5%) deceased. All SAEs were followed until resolution. However, at the time of dataset closure on 15 June 2021 (28 days after the last vaccination), 45 SAEs (32.6%) were still ongoing.

Table 2

Serious adverse events by Medical Dictionary for Regulatory Activities (MedDRA) system organ class and preferred adverse event term (n = 138).

System organ class and preferred adverse event term	N (%)	Incidence per 100,000 PY
Vascular disorders	37 (26.8%)	39.05 (28.30–53.90)
Pulmonary embolism	10	10.55 (5.68–19.62)
Ischaemic stroke	10	10.55 (5.68–19.62)
Deep vein thrombosis	4	4.22 (1.58–11.25)
Acute coronary syndrome	2	2.11 (0.53–8.44)
Hypertensive urgency	1	1.06 (0.15–7.49)
Intracranial hypertension	1	1.06 (0.15–7.49)
Leucocytoclastic vasculitis	1	1.06 (0.15–7.49)
Angiosarcoma	1	1.06 (0.15–7.49)
Retinal vein occlusion with macular haemorrhage	1	1.06 (0.15–7.49).
Subarachnoid haemorrhage	1	1.06 (0.15–7.49)
Cephalic vein thrombosis	1	1.06 (0.15–7.49)
Transient thrombosis of finger	1	1.06 (0.15–7.49)
Sagittal sinus thrombosis	1	1.06 (0.15–7.49)
Venous sinus and cortical venous thrombosis; subarachnoid and intraparietal haemorrhage	1	1.06 (0.15–7.49)
Nervous system disorders	31 (22.5%)	31.66 (22.14–45.29)
Headache	10	10.55 (5.68–19.62)
Bell palsy	5	5.28 (2.20–12.68)
Guillain-Barré syndrome	4	4.22 (1.58–11.25)
Paraesthesia in lower limbs	3	3.17 (1.02–9.82)
Ménière disease	2	2.11 (0.53–8.44)
Seizures	2	2.11 (0.53–8.44)
Transverse myelitis	2	2.11 (0.53–8.44)
Chronic fatigue syndrome exacerbation	1	1.06 (0.15–7.49)
Fibromuscular dysplasia	1	1.06 (0.15–7.49)
Functional neurological disorder	1	1.06 (0.15–7.49)
Immune system disorders	24 (17.4%)	24.28 (16.13–36.53)
Allergic reaction requiring hospitalisation	9	8.44 (4.22–16.88)
Severe reactogenicity symptoms requiring hospitalisation	6	6.33 (2.85–14.10)
Anaphylaxis	4	4.22 (1.58–11.25)
Reactive arthritis	2	2.11 (0.53–8.44)
Immune thrombocytopenic purpura	2	2.11 (0.53–8.44)
DRESS (related to NSAID use)	1	1.06 (0.15–7.49)
Multi-system symptoms	1	1.06 (0.15–7.49)
Infections and infestations	19 (13.8%)	20.05 (12.79–31.44)
Non-COVID-19 pneumonia	5	5.28 (2.20–12.68)
Acute appendicitis	3	3.17 (1.02–9.82)
Meningitis	2	2.11 (0.53–8.44)
Tuberculosis	2	2.11 (0.53–8.44)
Respiratory tract infection	2	2.11 (0.53–8.44)
Acute bronchitis	1	1.06 (0.15–7.49)
Pyelonephritis	1	1.06 (0.15–7.49)
Toxoplasmosis/tuberculoma	1	1.06 (0.15–7.49)
Interstitial pneumonitis	1	1.06 (0.15–7.49)
Mesenteric lymphadenitis	1	1.06 (0.15–7.49)
Musculoskeletal and connective tissue disorders	5 (3.6%)	5.28 (2.20–12.68)
Backache	1	1.06 (0.15–7.49)
Knee fracture dislocation	1	1.06 (0.15–7.49)
Disc prolapse with transient paralysis	1	1.06 (0.15–7.49)
Rhabdomyolysis	1	1.06 (0.15–7.49)
Transient myositis	1	1.06 (0.15–7.49)
Metabolism and nutritional disorders	4 (3.1%)	4.22 (1.58–11.25)
Hypoglycaemia	1	1.06 (0.15–7.49)
Hypoglycaemia and pneumonia	1	1.06 (0.15–7.49)
New-onset diabetes mellitus	1	1.06 (0.15–7.49)
Diabetic ketoacidosis	1	1.06 (0.15–7.49)
Gastrointestinal disorders	3 (2.2%)	3.17 (1.02–9.82)
Acute pancreatitis	1	1.06 (0.15–7.49)
Diarrhoea and vomiting	1	1.06 (0.15–7.49)
Haematemesis with per rectum blood clots	1	1.06 (0.15–7.49)
Respiratory disorders	2 (1.5%)	2.11 (0.53–8.44)
Acute asthma exacerbation	1	1.06 (0.15–7.49)
Chronic bronchitis	1	1.06 (0.15–7.49)
Hepatobiliary disorders	2 (1.5%)	2.11 (0.53–8.44)
Portal hypertension with upper GI bleeding	1	1.06 (0.15–7.49)
Liver dysfunction	1	1.06 (0.15–7.49)
Blood and lymphatic disorders	2 (1.5%)	2.11 (0.53–8.44)
Anaemia	1	1.06 (0.15–7.49)
Injury, poisoning, and procedural complications	1 (0.7%)	1.06 (0.15–7.49)
Injection site swelling
Psychiatric disorders	1 (0.7%	1.06 (0.15–7.49)
Major depressive episode
Renal and urinary disorders	1 (0.7%	1.06 (0.15–7.49)
Acute kidney injury
Cardiac disorders	1 (0.7%	1.06 (0.15–7.49)
Myocarditis recurrence
Unclassified	5 (3.6%)	5.28 (2.20–12.68)

DRESS, drug reaction with eosinophilia and systemic symptoms; GI, gastrointestinal; NSAID, non-steroidal anti-inflammatory drug; PY, person-years.

The most common vascular disorders were pulmonary embolism (n = 10, 10.6 per 100,000 person years, 95% CI 5.7–19.6) and ischaemic strokes (n = 10, 10.6 per 100,000 person years, 95% CI 5.7–19.6), followed by deep vein thrombosis (n = 4, 4.2 per 100,000 person-years, 95% CI 1.6–11.3). Three participants had both pulmonary embolism and deep vein thrombosis. There were 2 cases classified as TTS. The first case was a woman in the age group 46–50 years presenting with pulmonary embolism, thrombocytopenia, and positive anti-platelet factor 4 antibody assay 9 days after vaccination. She had a history of contraceptive injection use and underlying chronic respiratory and neurological conditions, and was being investigated for an autoimmune disorder. The second case was a woman in the age group 18–30 years who was admitted to hospital unconscious after experiencing a severe headache, restlessness, and confusion from 33 days after vaccination. A CT brain scan with venogram was in keeping with superior sagittal sinus thrombosis. Anti-platelet factor 4 antibody assay was negative, and she had marginally low platelets. She was a current smoker but had no other significant medical history. Both participants recovered.

Most events affecting the nervous system were complaints of headaches or migraines resulting in hospital admissions (n = 8). Five cases of Bell palsy (5.3 per 100,000 person-years, 95% CI 2.2–12.7) were reported between 1 and 42 days after vaccination; 2 men (age group 31–45 years) developed GBS about 2 weeks after vaccination, and 2 women (age group 46–55 years) developed GBS 16 and 17 days after vaccination (4.2 per 100,000 person-years, 95% CI 1.6–11.3). All participants were recovering at study end. Four cases were adjudicated as anaphylaxis by 2 physicians. All anaphylaxis cases had previous occurrence of drug- or vaccine-associated anaphylaxis and recovered fully. There was 1 case of myocarditis in a woman with previous myocarditis, which had settled prior to vaccination. She was receiving care at study end.

Table 3 summarises the disproportionality analysis that compares the occurred versus expected incidence ratio for SAEs of concern. Out of the SAEs examined, TTS seemed to occur at a rate greater than the baseline comparison, although the 95% confidence interval was wide and crossed 1 (O/E ratio 2.4, 95% CI 0.3–8.7), and GBS occurred at a rate greater than the baseline comparison population (O/E ratio 5.1, 95% CI 1.4–13.0). For the other SAEs, namely, ischaemic stroke, pulmonary embolism (non-TTS), deep vein thrombosis, acute coronary syndrome, Bell palsy, transverse myelitis, seizures, and myocarditis, the O/E ratio was less than 1. S2 Table presents the disproportionality analysis for SAEs occurring within 28 days of vaccination, and S1 Fig illustrates the frequency of SAE reporting from day of vaccination. As expected, there was a drop off in events when the analysis was restricted to 28 days post-vaccination, but there was not a significant change in the observed versus expected ratios of SAEs.

Table 3

Observed versus expected (O/E) analysis of selected serious adverse events.

Adverse event	Observed count	Observed incidence rate per 100,000 PY (95% CI)	Expected count	Expected incidence rate per 100,000 PY*	O/E ratio (95% CI)
Vascular disorders
Ischaemic stroke	10	10.55 (5.68–19.62)	102.89	108.60 [15]	0.10 (0.05–0.18)
Pulmonary embolism	10	10.55 (5.68–19.62)	21.09	22.26 (S4 Appendix)	0.47 (0.23–0.87)
Deep vein thrombosis	4	4.22 (1.58–11.25)	30.50	32.19 (S4 Appendix)	0.13 (0.04–0.34)
Acute coronary syndrome	2	2.11 (0.53–8.44)	214.12	226.00 [16]	0.01 (0.00–0.03)
Thrombosis with thrombocytopenia syndrome	2	2.11 (0.53–8.44)	0.83	0.88 [18]	2.40 (0.29–8.66)
Neurological disorders
Bell palsy	5	5.28 (2.20–12.68)	21.32	22.50 [17]	0.23 (0.08–0.55)
Guillain-Barré syndrome	4	4.22 (1.58–11.25)	0.79	0.83 [17]	5.09 (1.39–13.02)
Transverse myelitis	2	2.11 (0.53–8.44)	28.14	29.70 [17]	0.08 (0.01–0.27)
Seizure	2	2.11 (0.53–8.44)	69.45	73.30[17]	0.03 (0.00–0.10)
Cardiac disorders
Myocarditis	1	1.06 (0.15–7.49)	20.84	22.00 [17]	0.05 (0.00–0.27)

The O/E analysis compares the observed and expected numbers of cases. This may be expressed as the O/E ratio (observed incidence divided by expected incidence). The rates are for adults (males and females combined) and are not stratified by age group. Where a range is given in the literature on incidence, the mid-point was used. PY, person-years.

*Value followed by reference to literature from which the background incidence was derived.

A total of 157 (1.5% of participants reporting an AE) non-COVID-19-related deaths (167 per 100,000 person-years) were identified via the active linkage system with the national population registry. Of these deaths, cause of death was adjudicated for 67/157 (42.7%), and ascertainment continues for the remainder. Thirty-eight percent (n = 60) were male, median age was 48 years (IQR 40–57), 42 deaths (26.8%) were reported as having non-natural causes, 57% (n = 90) had at least 1 comorbidity; comorbidities reported were as follows: hypertension (n = 48, 30.6%), diabetes (n = 32, 20.4%), HIV (n = 20, 12.7%), heart disease (n = 9, 5.7%), and cancer (n = 1, 1.7%). Adjudicated causes of death included metastatic cancer (n = 18), HIV/AIDS-related deaths (n = 15), motor vehicle accidents (n = 11), homicide (n = 7), pulmonary embolism (n = 5), myocardial infarction (n = 4), cerebrovascular accident/stroke (n = 3), non-COVID-19 pneumonia (n = 3), intracerebral bleeding (n = 3), bleeding peptic ulcer/upper gastrointestinal tract bleeding (n = 3), suicide (n = 2), and status epilepticus (n = 2). Other causes are shown in S3 Table.

Nineteen deaths occurred within 28 days after vaccination. Causes were motor vehicle accident (3), upper gastrointestinal tract bleeding (3), homicide (2), HIV/AIDS-related deaths (2), and 1 each of pulmonary embolism, metastatic pancreatic cancer, drowning, dilated cardiomyopathy, renal failure, myelodysplastic syndrome, status epilepticus, suicide, and death after aortic valve and bypass surgery. A woman (age group 18–30 years) with a history of hypertension post-delivery presented 20 days after vaccination to her physician with jaundice and anuria. She then developed confusion, renal failure, and haemolysis requiring dialysis and fresh frozen plasma transfusion. She died after transfer to an intensive care unit. Investigations were in keeping thrombotic thrombocytopenic purpura (TTP). She was HIV negative, and no other triggers could be identified. Assessment of the event using the World Health Organization causality assessment tool classified this as an indeterminate temporal relationship with insufficient evidence for attribution to the vaccine [18]. In the absence of a clear alternative cause, the safety team deemed it plausible that the vaccine could have exacerbated this event in a patient with a predisposition to TTP.

Finally, we compared age-standardised mortality rates in the Sisonke study with projected background population mortality rates in South Africa as per the 2018 Medical Research Council Rapid Mortality Surveillance Report [19] and pre-COVID-19 local employee group life assurance data for a similar age-structured working population. The mortality rate in the Sisonke study was similar to the working population mortality data with similar ages, and well below that of the overall population mortality rate (Fig 2).

Fig 2

Age-standardised mortality rates by sex in the Sisonke study compared to 2018 South Africa mortality rates and working population mortality rates.

Age-standardised mortality rates for projected background population in South Africa as per the 2018 Medical Research Council Rapid Mortality Surveillance Report (RMS2018) [19] and pre-COVID-19 local employee group life assurance data (Group assured) for a similar age-structured working population. LL, 95% confidence interval lower limit; UL, 95% confidence interval upper limit.

Discussion

The Sisonke study, a large single-arm, open-label phase 3b implementation study, aimed to assess the safety and effectiveness of the single-dose Ad26.COV2.S vaccine among almost half a million HCWs in South Africa. A previous study of this vaccine supported its effectiveness against severe COVID-19 disease and COVID-19-related death after vaccination, and against both the beta and delta variants [20]. With over 10,000 AE reports, to our knowledge, this was the largest safety analysis of the Ad26.COV2.S vaccine from a low- or middle-income country. As observed in phase 3 trials, similar patterns of AEs were found and were mostly expected reactogenicity signs and symptoms. Furthermore, most SAEs were rare and occurred below expected rates. However, we did observe very rare events of TTS and GBS in this study at apparently higher than expected rates, though confidence intervals for these estimates were wide. Nevertheless, overall, this study provides additional real-world evidence that the vaccine is safe and well tolerated, supporting its continued use in this setting.

AEs were more often reported by women than men. While this observation may illustrate a stronger immune response in females compared to males as seen for other vaccines [21-23], behavioural factors such as reduced reporting among men may have also played a role, though these factors were not measured. The prevalence of reported AEs decreased with increasing age. A number of studies show that vaccine-related AEs and reactogenicity are less prevalent in older people due to the waning of innate immune defence mechanisms; lower systemic levels of IL-6, IL-10, and C-reactive protein; and lower neutralising antibody titres after vaccination as compared to younger individuals [24-26]. Individuals reporting previous COVID-19 infection seemed to have higher AE reporting rates. Some studies suggest that there is increased immunogenicity in the setting of past infection, leading to higher antibody titres and therefore higher reactogenicity rates [27,28].

TTS and GBS occurred at very low rates in this study; however, the disproportionality analysis showed a higher event rate than expected in the population. After 12.6 million doses of the Ad26.CoV.2 vaccine were administered in the US, 38 confirmed TTS cases and 98 GBS cases were reported [10]. Based on these data, estimates illustrate a clear advantage of vaccination despite these rare AE occurrences. For example, among women aged 30–49 years in the US, for every 6–7 cases of GBS or 8–10 cases of TTS, 10,100 COVID-19 cases, 900 hospitalisations, 140 intensive care unit admissions, and 20 deaths were prevented [10].

While the risk–benefit balance clearly favours vaccination, this study highlights the importance of ongoing safety monitoring in population-based vaccination programmes to enable continued risk–benefit assessment. The Sisonke study shows that additional surveillance, heightened awareness, and development of protocols for the management of potential clinical complications after vaccination help identify and manage possible cases early and appropriately. For example, the 2 cases of TTS were successfully managed with the support of the protocol safety review team, and both participants recovered. It is crucial that such cases are identified promptly to enable successful management. Local clinical recommendations for management of TTS were developed and implemented [29].

The Sisonke study had some limitations. First, the surveillance system was primarily passive, relying on self-reporting; thus, some AEs may have gone unreported. It is likely that the system was better suited to detect SAEs than milder AEs, which participants may have ignored rather than reported. Second, as active contact with participants was continued up to 2 weeks post-vaccination, it is probable that SAEs other than deaths and COVID-19 events were more likely to be reported during this period, and there may have been some underestimation of SAEs that occurred later. The active linkage of the unique identifier in the EVDS with deaths in the national population registry and with COVID-19 events in the COVID-19 laboratory system ensured identification of nearly all possible deaths and COVID-19 events in the study. Third, considering the large number of participants in the study, not all self-reported AEs could be verified, and only SAEs and AEs of medical concern were investigated further. Fourth, the disproportionality analysis should be interpreted with caution given the uncertainties in both the observed and expected event rates, variable follow-up time, non–South African reference data for some groups, and potential differences in age–sex distributions between the Sisonke study and reference data. However, while disproportionality analysis in the context of safety signal detection is mainly exploratory, it has the utility of identifying potentially important associations between AEs and vaccines. In this study, the analysis confirmed current reports of the safety risk of the Ad26.COV2.S vaccine with respect to TTS and GBS [10]. Lastly, it is also important to note that without a placebo group, open-label, single-arm studies are subject to measurement bias with the potential of overreporting of AEs, and hence some caution is needed in interpreting safety data. No other safety concerns were found in this study [10,11].

Overall, the Sisonke study did not identify excess deaths in the vaccinated population compared to the general population. Mortality rates were comparable to a similar adult working population from 2018. This report illustrates the importance of accurate national mortality surveillance, especially in settings where vaccine hesitancy is driven by non-scientific and inaccurate reports in communities and through social media. The Sisonke study results are also a strong reminder that South Africa faces a large burden of disease [30]. While cancer was the most common cause of death during the study period, highlighting the urgent need for specialist oncology services, it is concerning that among HCWs advanced HIV and tuberculosis remain common causes of death. It shows that despite gains in access to HIV testing and treatment, HIV and tuberculosis care require further improvement. Local data highlight that the COVID-19 epidemic heavily impacted HIV testing and treatment initiations [31]. Motor vehicle accidents and homicides were also common causes of death, a reflection of the injury and trauma burden in South Africa. One death was related to TTP, which has previously been reported after Ad26.COV2.S vaccination and warrants further evaluation in other studies [32].

In conclusion, this study affirms that the single-dose Ad26.COV2.S vaccine is safe and well tolerated when administered to adults in South Africa. Few SAEs were observed, and they were successfully managed with prompt identification. The Sisonke study underscores the value of setting up robust pharmacovigilance systems for prompt identification, evaluation, and reporting of AEs to enable continued assessment of the risk–benefit profiles of COVID-19 vaccines. This has the potential to improve public confidence in vaccine safety and reduce vaccine hesitancy.

Sisonke study protocol.

(PDF)

Click here for additional data file.

Sisonke study adverse event case report form.

(PDF)

Click here for additional data file.

Sisonke study statistical analysis plan.

(PDF)

Click here for additional data file.

Background incidence rates for pulmonary embolism and deep vein thrombosis.

(DOCX)

Click here for additional data file.

Safety manuscript tables DO file.

(DO)

Click here for additional data file.

CONSORT checklist.

(DOC)

Click here for additional data file.

Frequency of serious adverse event reports from time of vaccination.

(TIF)

Click here for additional data file.

Twenty most common non-reactogenicity adverse events reported.

(DOCX)

Click here for additional data file.

Observed versus expected analysis of selected serious adverse events within 28 days of vaccination.

(DOCX)

Click here for additional data file.

Mortality in the Sisonke study.

(DOCX)

Click here for additional data file.

1 Dec 2021

Dear Dr Takuva,

Thank you for submitting your manuscript entitled "Safety of the single-dose Ad26.CoV2.S vaccine among healthcare workers in the phase 3b Sisonke study in South Africa" for consideration by PLOS Medicine.

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23 Feb 2022

Dear Dr. Takuva,

Thank you very much for submitting your manuscript "Safety of the single-dose Ad26.CoV2.S vaccine among healthcare workers in the phase 3b Sisonke study in South Africa" (PMEDICINE-D-21-04884R1) for consideration at PLOS Medicine.

Your paper was evaluated by a senior editor and discussed among all the editors here. It was also discussed with an academic editor with relevant expertise, and sent to three independent reviewers, including a statistical reviewer. The reviews are appended at the bottom of this email and any accompanying reviewer attachments can be seen via the link below:

[LINK]

In light of these reviews, I am afraid that we will not be able to accept the manuscript for publication in the journal in its current form, but we would like to consider a revised version that addresses the reviewers' and editors' comments. Obviously we cannot make any decision about publication until we have seen the revised manuscript and your response, and we plan to seek re-review by one or more of the reviewers.

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plosmedicine.org

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Requests from the editors:

1. Title: Please revise your title according to PLOS Medicine's style. Your title must be nondeclarative and not a question. It should begin with main concept if possible. "Effect of" should be used only if causality can be inferred, i.e., for an RCT. Please place the study design ("A randomized controlled trial," "A retrospective study," "A modelling study," etc.) in the subtitle (ie, after a colon).

2. Financial disclosure: Thank you for indicating that the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Please add to your statement in the Financial Disclosure section of the submission form: the full names of each funder, specific grant numbers, the initials of authors who received each award, and URLs to sponsors’ websites.

3. Main text: Please provide line numbers running continuously through the manuscript with the revised version.

4. Abstract: Please structure your abstract using the PLOS Medicine headings (Background, Methods and Findings, Conclusions) Please combine the Methods and Findings sections into one section, “Methods and findings”.

5. Abstract: Background: Provide a sentence or two of context of why the study is important. The final sentence should clearly state the study question.

6. Abstract: Methods and Findings: Please describe the study design, and population and setting.

7. Abstract: Methods and Findings: For the following two statements, please provide the numbers/percentages associated with these reported AEs: “The commonest reactogenicity events were headache and body aches, followed by injection site pain and fever…” and “Serious AEs and AEs of special interest including vascular and nervous system events, immune system disorders and deaths occurred at lower than the

expected population rates.”

8. Abstract: Methods and Findings: In the last sentence of the Abstract Methods and Findings section, please describe the main limitation(s) of the study's methodology.

9. Abstract: Conclusions: Please address the study implications without overreaching what can be concluded from the data; the phrase "In this study, we observed ..." may be useful.

10. Author summary: At this stage, we ask that you include a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract. Please see our author guidelines for more information: https://journals.plos.org/plosmedicine/s/revising-your-manuscript#loc-author-summary

11. Main text: Please use square brackets for in-text citations of references, placed before the sentence punctuation, for example [1,2].

12. Background: Please title this section of the main text “Introduction” and please conclude the Introduction with a clear description of the study question or hypothesis.

13. Methods: Please report your study according to the relevant guideline, which can be found here: http://www.equator-network.org/

Please ensure that the study is reported according to the CONSORT guidelines, and please complete the CONSORT checklist and ensure that all components of CONSORT are present in the manuscript.

When completing the checklist, please use section and paragraph numbers, rather than page numbers.

Please add the following statement, or similar, to the Methods: "This study is reported as per the Consolidated Standards of Reporting Trials (CONSORT) guideline (S1 Checklist)."

14. Methods: Please provide additional description of inclusion and exclusion criteria as indicated in more detail online (PACTR/clinical trials.gov registries). Please explicitly how participants were recruited, and mention that informed written consent of participants was obtained. Please comment on whether it is possible there were individuals included who had received other vaccines to prevent SARS-CoV-2 infection. Please mention/provide numbers to inform if there were eligible individuals who were contacted but declined to participate. Please include a participant flow diagram.

15. Methods: Please include the study protocol document and the analysis plan, with any amendments, as Supporting Information to be published with the manuscript if accepted.

16. Methods: Please include a copy of the electronic case report form designed for the study as a supporting information document. Please provide additional information for the verification of SAEs and AEs of medical concern, and for spontaneous reports via unsolicited HCW communication.

17. Methods: Please provide a list of those events considered as serious AEs/AEs of special interest.

18. Methods: Missing data have the potential to introduce bias in your study. Please explain how you have dealt with missing data.

19. Methods: Please describe how individual SAEs were selected for this analysis: “For selected SAEs, disproportionality analysis was conducted…”

20. Methods: Statistical analysis: “Participants reporting and not

reporting AEs were compared by baseline characteristics.” Please describe the collection of baseline characteristic data, and please describe how these data were incorporated into analyses. Please explain the categorization for age.

21. Methods: Statistical analysis: “Available background incidence rates were used including a medically insured population in South Africa (pulmonary embolism and deep venous thrombosis), Tanzanian population-based cohort study (neurological events such as stroke)

and European population databases.(10, 15–18).” Please provide more information, including how expected count/incidence were obtained for Table 3.

22. Results: Please clarify here if all post-vaccination follow up contacts provided additional data (on whether AEs resolved or not), and if not, please mention numbers lost to follow up. “Follow up at day seven post vaccination indicated that 92% of participants reporting AEs had either completely recovered or were recovering. The remaining 8% of participants were contacted by the safety team and, if required, referred for care.”

23. Results: Please present numerators and denominators for percentages, at least in the Tables if not in the main text of the Results [not necessarily each time they're mentioned].

24. Results: “One in five (19%) AEs were not consistent classified as reactogenicity events (Supplementary Table 1).” Please clarify in the text what is meant here (for example, if these were classified as serious adverse events).

25. Results/ Figure 2: Please provide additional details regarding the comparison with the local employee group life assurance data.

26. Discussion: Please be sure to present and organize the Discussion as follows: a short, clear summary of the article's findings; what the study adds to existing research and where and why the results may differ from previous research; strengths and limitations of the study; implications and next steps for research, clinical practice, and/or public policy; one-paragraph conclusion.

Comments from the reviewers:

Reviewer #1: See attachment

Michael Dewey

Reviewer #2: S. Takuva et al., reported here the results of an open label implementation study that assess the safety (and the effectiveness) of one shot of Ad26.CoV2.S vaccine of janssen among Healthcare workers (HCWs) in South Africa. This manuscript is focused on the presentattion of safety data.only.

It is a very impresssive implementation study that included in real life conditions an half of a million of HCWs.

The manuscript is well written and easy to read.

The introduction is concise and presented well the situation.

I have few comments:

-in the methods section, (p 5) please provide details about the exclusion of pregnant women ( there is a pregnancy test performed? what could be challenging in study of this size; if not just precise that women with a known pregnancy were excluded).

-in the results section, since the numerators are not notified in the text, please propose to the reader to see in the table 1, as it is only the percentage is available ("the majority were women (74.9%)"). In the section "SAE" of the results section page 8, it is written "114 reported by women and 25 by men" 114+25= 139 , only 138 SAE s excluding death were reported, please change. In this same paragraph, please specify at which time after vaccination the SAEs outcome were evaluated?. For the 9 death (6.5%), I suggest to precise that it represented 9/10,279 (0.09%) of people that declared an AE.

-references in the reference list are sometime not completed

This work is very interesting and indeed shows that, including in low and middle income countries, surveillance at large scale is possible, what is crucial to achieve confidence in vaccines.

Reviewer #3: I thoroughly enjoyed reading the "Safety of the single-dose Ad26.CoV2.S vaccine among healthcare workers in the phase 3b Sisonke study in South Africa" by Takuva et al. It was clearly written in excellent English, with a format and logic that was cohesive and easy to follow. Their sampling and selection criteria is clear, and their analysis follows standard methods. The reporting of their results is clear, and in their discussion and limitations they follow a logical expansion of the topic. The only suggestions that I have to improve the quality of their excellent work are as follows:

1- Given the high percentage of females in the study, a brief explanation of whether they over sampled for females would be clarifying.

2- In Tables 2 and 3, if there could be an additional column which indicates the incidence of adverse events in other large studies of the safety Ad26.CoV2.S (perhaps Ref. 7 & 8) it would allow for rapid comparison of results.

3- Several minor typo mistakes, which fall under the editorial corrections.

Any attachments provided with reviews can be seen via the following link:

[LINK]

Submitted filename: takuva.pdf

Click here for additional data file.

8 Apr 2022

Submitted filename: Response to Reviewers_ Sisonke PLOS Med.docx

Click here for additional data file.

5 May 2022

Dear Dr. Takuva,

Thank you very much for re-submitting your manuscript "Safety evaluation of the single-dose Ad26.COV2.S vaccine among healthcare workers in the Sisonke study in South Africa: a phase 3b implementation trial" (PMEDICINE-D-21-04884R2) for review by PLOS Medicine.

I have discussed the paper with my colleagues and the academic editor and it was also seen again by two reviewers. Provided that the remaining reviewer comments, and editorial and production issues are dealt with we are planning to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Any accompanying reviewer attachments can be seen via the link below. Please take these into account before resubmitting your manuscript:

[LINK]

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To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

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If you have any questions in the meantime, please contact me or the journal staff on plosmedicine@plos.org.

We look forward to receiving the revised manuscript by May 12 2022 11:59PM.

Sincerely,

Caitlin Moyer, Ph.D.

Associate Editor

PLOS Medicine

plosmedicine.org

------------------------------------------------------------

Requests from Editors:

1. Response to Reviewers: Please completely address the remaining point of Reviewer 1 by providing additional details on effects on participants for adverse events reported in Supporting Information Table 1.

2. Title: Please capitalize the first letter of the first word of the subtitle, and please update this in both the manuscript text and the manuscript submission system: “Safety evaluation of the single-dose Ad26.COV2.S vaccine among healthcare workers in the Sisonke study in South Africa: A phase 3b implementation trial”

3. Data availability statement: The Data Availability Statement (DAS) requires revision. If the data are owned by a third party (The National Department of Health, in this case), please note whether the data may be made available upon request or application, and state the owner of the data set and contact information for data requests (as you have done already: Office of the Director-General, E-mail: DG@health.gov.za).

We request that the analytical code be made available, without restrictions on access, in a public repository or included as Supporting Information at the time of article publication, provided it is legal and ethical to do so. We note that the Protocol and Analysis Plan are included as supporting information files already, and these files do not need to be mentioned here.

Please update this statement with information needed to access the analytical code. Please note that a study author may not serve as the point of contact for access. Please see the policy at

http://journals.plos.org/plosmedicine/s/data-availability

and FAQs at

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4. Abstract: Methods and Findings: Please provide the trial registration information in the Abstract.

5. Abstract: Methods and Findings: Please clarify “...among all eligible HCWs in South Africa registered in the national Electronic Vaccination Data System (EVDS)…” or similar.

6. Abstract: Methods and Findings: At line 77, the number of serious AEs is reported as 139, and at line 85, this is reported as 138.

7. Abstract: Conclusions: Line 91-93: Given the finding in Table 3 for TTS and GBS, it may be more accurate to phrase this as “most SAEs occurred below expected rates.”

8. Author summary: Line 108-111: Please revise to: “The majority of adverse events reported…” and please revise the following sentence to “ Most serious adverse events…”

9. Methods: Please add the following statement, or similar, to the Methods: "This study is reported as per the Consolidated Standards of Reporting Trials (CONSORT) guideline (S1 Checklist)."

10. Methods: Line 173-174: Please reference the copy of the study protocol included as a supporting information file, and refer to it here (Supplementary Appendix 2). Please cite the online version of the protocol in the reference list, rather than including a web link here.

11. Methods: Statistical analysis: Line 239: Please mention If a prospective analysis plan was used in designing the study, for example, Supplementary Appendix 3, and please reference that file here. Please describe in the text if any changes to the analysis plan were made, and when.

12. Methods: Line 241-242: “Participants reporting and not reporting AEs were compared by baseline characteristics.” Please provide more details of this analysis.

13. Methods: Line 257-258: “COVID-related deaths were excluded in this report and published in a separate effectiveness report.” Please provide a reference to the publication if these data have been published.

14. Results: Line 266-268: We suggest rephrasing to: “...(all 1 250 000 HCWs in the country were invited to participate).” or similar.

15. Results: Line 279: Please change “less” to “fewer” AEs.

16. Results: Line 292-296: “Follow up at day seven post vaccination indicated that 92% of participants reporting AEs had either completely recovered or were recovering. The remaining 8% of participants were contacted by the safety team. Attempts were made to contact all these participants individually by the safety team (three telephonic calls at least one day apart). Those that were contactable were captured and if indicated, referred for further care.” Please include numbers in addition to reporting percentages here. Please clarify what is meant by “Those that were contactable were captured…” and please re-phrase this if possible.

17. Results: Line 296-297: “One in five (19%) AEs were not consistent classified as reactogenicity events” Please provide the actual number of AEs not classified as reactogenicity events.

18. Results: Line 307-308: “SAE outcomes were: 48 (34.8%) recovered, 36 (26.1%) recovering, 45 (32.6%) ongoing and 9 (6.5%) deceased.” Please mention the duration of follow up here, in light of the 32% of reports classified as ongoing and the following sentence mentioning events were followed up until resolution.

19. Discussion: Line 392-394: We suggest phrasing this as: “A previous study of this vaccine supported its effectiveness against severe COVID-19 disease and COVID-19-related death after vaccination, and against both beta and delta variants [20].

20. Discussion: Line 397-398: We suggest revising to: “Furthermore, most SAEs were rare and occurred below expected rates. However, we did observe very rare events of TTS and GBS in this study at apparently higher than expected rates, though confidence intervals for these estimates were wide.” or similar.

21. Discussion: Line 405: We suggest “ was lower with increasing age” instead of “reduced” here.

22. References: Please check the formatting of each reference, and please use the "Vancouver" style for reference formatting, and see our website for other reference guidelines https://journals.plos.org/plosmedicine/s/submission-guidelines#loc-references

23. When providing a DOI (e.g. for Reference 3), please give both the label and full DOI at the end of the reference. Do not provide a shortened DOI or the URL.

Please remove “[Internet]” throughout.

Please update the citation information for any preprints (e.g. reference 16).

24. Table 1: Please present p values for all comparisons. Please define all abbreviations used (AEs, IQR, HIV) in the legend.

25. Table 2: Please define abbreviations used (PY, DRESS, GI) in the legend.

26. Table 3: Please define abbreviations used (PY) in the legend. In the legend, please clarify if this should be “The rates are for adults (males and females combined) and are not stratified by age-group.”

27. Figure 1: Please provide a descriptive legend for the graph.

28. Figure 2: Please describe “UL” and “LL” as well as indicating 2018 Medical Research Council Rapid Mortality Surveillance Report (RMS2018) and pre-COVID-19 local employee group life assurance data (Group assured) in the legend.

29. CONSORT Checklist: Thank you for including the checklist. Please revise the checklist to refer to locations within the text with section and paragraph numbers (e.g. Methods, paragraph 1). Please do not refer to page numbers.

30. Supplementary Table 1: Please spell out the abbreviation “BP” in the legend.

31. Supplementary Table 2: Please define abbreviations (PY, CI, O/E) in the legend.

32. Supplementary Table 3: Please define abbreviations (IQR, HIV) in the legend.

33. Supplementary Appendix 2: Please note that pages 2-6 contain contact information (addresses/phone numbers/email addresses), please remove this information if it should not be made publicly available.

Comments from Reviewers:

Reviewer #1: The authors have addressed my points but there remains one minor issue outstanding. I asked for Supplementary Table 1 to give us more details about these other adverse events broken down by the effect on the participant (unable to perform usual activities, hospitalised). I perhaps was not clear enough as the authors' response just outlines what response the clinical and research team made to the events. This is valuable to know but not what I was asking for.

Michael Dewey

Reviewer #2: Authors correctly addressed the comments of reviewers

Any attachments provided with reviews can be seen via the following link:

[LINK]

12 May 2022

Submitted filename: Responses to reviewers.RV2.docx

Click here for additional data file.

16 May 2022

Dear Dr. Takuva,

Thank you very much for re-submitting your manuscript "Safety evaluation of the single-dose Ad26.COV2.S vaccine among healthcare workers in the Sisonke study in South Africa: A phase 3b implementation trial" (PMEDICINE-D-21-04884R3) for review by PLOS Medicine.

Provided the remaining editorial and production issues are dealt with we are planning to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Any accompanying reviewer attachments can be seen via the link below. Please take these into account before resubmitting your manuscript:

[LINK]

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

In revising the manuscript for further consideration here, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.

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To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

Please note, when your manuscript is accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you've already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosmedicine@plos.org.

If you have any questions in the meantime, please contact me or the journal staff on plosmedicine@plos.org.

We look forward to receiving the revised manuscript by May 18 2022 11:59PM.

Sincerely,

Caitlin Moyer, Ph.D.

Associate Editor

PLOS Medicine

plosmedicine.org

------------------------------------------------------------

Requests from Editors:

1. Line 69-70: Please move the clinical trial information to the end of the abstract. “ClinicalTrials.gov number NCT04838795; Pan-African Clinical Trials registry number PACTR202102855526180.”

2. Abstract: Line 86-87: Please revise to “Most serious AEs and AEs of special interest (n=138) occurred at lower than the expected population rates.”

3. Line 90: Please spell out “SAE” at first use in the abstract.

4. Author summary: Line 116: Please re-phrase or clarify what is meant by “the large roll-out products.”

5. Line 299-300: Please remove the extraneous text from this paragraph. “One in five (19%, 2,109) AEs were not consistently classified as reactogenicity events” Please provide the actual number of AEs not classified as reactogenicity events.”

6. Table 3, Supplementary Table 2: In the table legends, please define the numbers in parentheses in the expected incidence rate columns.

7. Reference list: Please remove reference 17 or replace with another reference that is accessible. The reference list may not include unavailable and unpublished work. If relevant you may include the information or data as supplementary material or deposit the data in a publicly available database.

8. Page 17: Please also list the CONSORT Checklist, Protocol, CRF, and SAP as supporting information files.

9. Analytical code: Please rename the supporting information file with the analytical code with a more clear name and a title, and please list it at the end of the manuscript with the other supporting information files. Please refer to this file at the appropriate place in the Methods section.

Any attachments provided with reviews can be seen via the following link:

[LINK]

17 May 2022

Submitted filename: Responses to reviewers.RV3.docx

Click here for additional data file.

19 May 2022

Dear Dr Takuva,

On behalf of my colleagues and the Academic Editor, Amitabh Bipin Suthar, I am pleased to inform you that we have agreed to publish your manuscript "Safety evaluation of the single-dose Ad26.COV2.S vaccine among healthcare workers in the Sisonke study in South Africa: A phase 3b implementation trial" (PMEDICINE-D-21-04884R4) in PLOS Medicine.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Once you have received these formatting requests, please note that your manuscript will not be scheduled for publication until you have made the required changes.

In the meantime, please log into Editorial Manager at http://www.editorialmanager.com/pmedicine/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

Please also address the following editorial request:

Table 3 and Supporting Information Table 2: Please use square brackets to indicate references, for consistency with the main text. Please note “S4 Table” where applicable.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with medicinepress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Thank you again for submitting to PLOS Medicine. We look forward to publishing your paper.

Sincerely,

Caitlin Moyer, Ph.D.

Associate Editor

PLOS Medicine