Evaluation of Effectiveness and Safety of LC16m8 Mpox Vaccine in the Democratic Republic of Congo (DRC)

1. Community Engagement: The community mobilization will be carried out according to the national communication plan for the vaccination promotion. Study staff members will engage with religious leaders, medical professionals, heads of villages, and key opinion leaders to share information about the mpox disease surveillance, the availability of the vaccine, and the vaccination. Additionally, detailed information regarding the available mpox testing and treatment facilities that the community needs to visit if any person develops symptoms consistent with mpox or develops adverse events following vaccination will be provided in the community engagement activities. Special emphasis will be made in the community messaging to encourage all individuals with mpox-compatible symptoms regardless of the disease severity and vaccination status-to promptly seek testing and care. 2. Enhanced Health Facility-based Mpox Disease Surveillance: Health facility-based and enhanced mpox surveillance will be implemented to record mpox cases within the vaccination catchment area. The study will be done in 2 or more health zones identified as hot spots by the DRC government.

The healthcare facilities within the study catchment area will be equipped with mpox testing equipment \& supplies and the study staff will receive comprehensive training for the mpox case investigation, sample collection, and management. Pre-existing laboratories will be supported by the project to perform RT-PCR tests, and a subset of samples will be shipped to the reference microbiology laboratory at INRB headquarters in Kinshasa for quality control.

Patients presenting at a sentinel surveillance healthcare facility with suspected or probable mpox will be approached for enrollment. For patients presenting with skin lesions, a swab of the skin will be collected from all suspected/probable mpox cases for testing using RT-PCR. Out of the patients presenting with skin lesions, one hundred patients will be consecutively invited to provide oro-pharyngeal swab and saliva to assess the diagnostic accuracy of other samples compared to skin swab. For patients presenting without skin lesions, oro-pharyngeal swab will be taken. Each suspected case will be asked for contact information (telephone number and/or description of residence). After 3 weeks, study staff will seek to contact, by telephone and/or in-person visit, each suspected case who tested negative by RT-PCR at enrollment to assess whether or not the individual experienced additional symptoms and signs consistent with symptomatic mpox, particularly the development of skin lesions. The study staff will seek to collect skin or oropharyngeal swabs from initially test-negative patients who reported experiencing additional symptoms consistent with symptomatic mpox during the three-week period after enrollment and to test these samples by RT-PCR. 1. Laboratory Procedure: Real-time polymerase chain reaction (RT-PCR) for the presence of MPXV in skin swabs from all patients presenting with skin lesions suggestive of mpox will be performed for laboratory confirmation and diagnosis of mpox. For patients presenting without skin lesions, oro-pharyngeal swab will be tested using RT-PCR. The RT-PCR protocols for the detection of MPXV will be performed according to manufacturer's instructions, in line with WHO guidelines for the detection of MPXV. 2. Specimen Type, Collection, Transport and Storage: Skin lesion material includes swabs of lesion surface and/or exudate, or lesion crusts. Lesions will be swabbed vigorously to ensure adequate viral DNA is collected. Swabs will either be transported dry in capped tubes or placed in viral transport media (VTM). Whenever possible, specimens from two lesions should be collected in one single tube, preferably from different locations on the body. For patients with no skin lesions at presentation, oro-pharyngeal swabs will be taken. Samples will be stored at 2-8°C if processed within 72 hours. Samples that are shipped to reference laboratories will be stored at -20°C or lower. Longer-term storage (\>60 days) will be at -70°C. All specimens being transported should have appropriate triple packaging, labelling, and documentation. 3. Vaccination using LC16m8 Vaccine: As the study aims to generate real-world vaccine effectiveness by leveraging DRC's national mpox vaccination strategy, the vaccination will be conducted in 2 to 3 selected health zones/areas which meet the criteria for a hotspot as defined by the DRC vaccination strategy for targeted vaccine delivery.

Selection of health zones/area for the study where LC16m8 is expected to be offered to everyone one year or older, will be determined based on the latest mpox epidemiological data, operational feasibility, and existing research infrastructure in close consultation with DRC MoH, Institut National de Santé Publique (INSP), and local stakeholders. Due to on-going mpox outbreaks in multiple provinces in DRC, investigators will also ensure enough doses are allocated and delivered to selected health zones/areas to reach minimum coverage required for the study (i.e., 30% coverage). Details of the vaccination strategy will be determined in close collaboration with local public health authorities.

a. Vaccination Information Registration: As mpox vaccine is being offered as part of DRC's national mpox vaccination strategy, consent/assent procedure for the vaccination will be carried out according to the routine public health immunization program. Vaccination sites/centers will collect mpox vaccination registry as per routine practice as well and provide a vaccination card along with detailed instructions on how to securely retain it for future reference.

In addition to routine immunization practices, each person receiving LC16m8 vaccine in selected health zones/areas will be asked to provide biometric information (i.e., iris scanner). Study staff will collect informed consent/assent from vaccinees prior to collecting any biometric data. Collected biometric information along with mpox immunization records will be registered to a dedicated database (e.g., REDCap platform), which will be maintained by the INRB. In case vaccinees reject to provide any biometric information for the study purpose, only paper-based immunization registry will be collected as per routine practice.

For those who agreed to provide biometric information, vaccination records will be merged with provided biometric information. For paper-based registry, it will be entered into electronic vaccination database (i.e., REDCap platform managed by INRB). The vaccination database will include key personal identifiers, which will be used in an electronic "parsing tool," such as the one developed in-house by the International Vaccine Institute (IVI), which is a software capable of linking sociodemographic data of an individual to a vaccine registry database. Biometric information and/or "parsing tool" on electronic vaccination database will be used to correctly identify mpox vaccination history for those enrolled into the test-negative case-control study to enable calculation of VE estimates 4. Safety Monitoring Following Immunization with LC16m8 Vaccine: All participants receiving the LC16m8 vaccine will be observed for the first 30 minutes of vaccination as per the DRC's national vaccination strategy. The monitoring of the LC16m8 vaccine safety administered during vaccination will be assessed through:

1. A cohort event monitoring (CEM), an observational prospective cohort study: At enrollment/vaccination visit, participants will be consecutively invited to be enrolled in the safety subset until the sample size of 10,000 has been achieved. The enrollment aims to include a proportional number of participants from various age groups, including 1-5 years, 6-12 years and 13-17 years, with representation from both sexes. Enrolled vaccinees will be actively followed up through phone calls/home visits/Health facility visits for the SAEs and AESIs assessment at weekly intervals for the first month and then at monthly intervals until 12 weeks post vaccination. Vaccinees/parents/guardians will be advised to return to the sentinel health facility if any serious event occurs at any time. All adverse events including AESIs and SAEs will be assessed using CRFs adapted from existing tools from WHO protocol. All SAEs and AESIs will be assessed for relatedness, seriousness, expectedness, and outcome.

Participants will have the right to withdraw from the study at any time and for any reason. Withdrawn participants will not be replaced after the recruitment of participants has ended. Data collected from withdrawn participants will be used for study purposes. A participant will be considered as lost-to follow-up after three documented unsuccessful attempts have been made to contact the participant, by phone/home visit. Participants that are lost to follow-up will not be replaced after the enrollment is completed. 2. A passive surveillance at sentinel surveillance sites: Passive AEFI surveillance will be conducted at designated health facilities within the catchment area. Therefore, data on SAEs \& AESI will be collected through health facility records or voluntarily reporting of event(s) by the vaccinees/parents or guardians (self-reported data) and by healthcare workers. Medical records in these facilities will be regularly reviewed to detect any potential serious adverse events or AESIs associated with vaccination including adjudication of causality, relatedness, seriousness and temporal relationship with vaccination. Potential SAEs and AESI will be recorded in dedicated forms by a trained health professional. Vaccination data will be linked to the passive surveillance data using the same database/parsing tool used for vaccination ascertainment in the VE study.

Female study participants of childbearing age will be instructed to report to the study staff any pregnancy case which occurs within three months after receiving the vaccine. Pregnant women who are inadvertently exposed to the LC16m8 vaccine and women who become pregnant within 3 months of vaccination will be followed every three months until delivery or the end of pregnancy. The neonatal and maternal outcomes will be recorded based on records at the health facility. Pregnancy itself is not considered an AEFI, but any complications during pregnancy are to be considered as AEFI, and in some cases could be considered Serious AEFI. Spontaneous abortions, blighted ovum, fetal death, stillbirth, an elective termination for medical rationale and congenital anomalies reported in the baby are always considered as Serious AEFI, and the information should be provided using an appropriate Serious AEFI form.

SAE and AESIs will be actively monitored over 12 weeks after immunization in the safety cohort and throughout the study duration as part of passive surveillance. 5. LC16m8 Vaccine Effectiveness using the Test-Negative Design: An observational, prospective, health facility-based study, following the TND will be carried out in each participating site to assess the effectiveness of vaccination using the LC16m8 vaccine. In the TND study, the cases and controls are both seeking services for mpox diagnostic testing and management because of mpox-like illnesses - the cases being those testing positive for mpox using RT-PCR and the controls those testing negative . As such, the bias introduced by healthcare-seeking differences is controlled by implementing TND study for vaccine effectiveness (VE) evaluation by disease severity. 6. Statistical Considerations

1. Sample Size

* Sample Size for the VE Assessment: Vaccine effectiveness will be estimated using cases and controls assembled based on specific inclusion/exclusion and matching criteria. Detailed matching procedures will be described in the statistical analysis plan (SAP). Assuming a vaccine effect

• The percentage of participants with complete vaccination in different age strata of participants with RT-PCR-confirmed mpox disease compared with the percentage of participants with complete vaccination in those without mpox disease. [Time frame: At baseline (Day 0)]
• The percentage of participants with incomplete vaccination among participants with RT-PCR-confirmed mpox disease compared to the percentage of participants with incomplete vaccination among participants with negative mpox RT-PCR(overall&stratified by age [Time frame: At baseline (Day 0)]
• The percentage of participants with complete vaccination in those with RT-PCR-confirmed severe mpox disease compared to the percentage of participants with complete vaccination in those without mpox (overall & stratified by age). [Time frame: From baseline (Day 0) to severe outcome (death or hospitalization, assessed up to Day 42)]
• The percentage of participants with incomplete vaccination in those with RT-PCR-confirmed severe mpox disease compared to the percentage of incomplete vaccination among those without mpox disease (overall & stratified by age). [Time frame: From enrollment (Day 0) to severe outcome (death or hospitalization, assessed up to Day 42)]
• Evaluate the incidence of serious adverse events (SAEs) and adverse event of special interest (AESI) of the LC16m8 vaccine using a cohort event monitoring (CEM) in a defined cohort and passive surveillance in all vaccinees. [Time frame: Within 12 weeks following immunization in the CEM cohort and with 12 months in the rest of the vaccinees (not part of the CEM cohort)]