Assessment of Myocarditis After Replication-Deficient Smallpox Immunization

Receipt of the novel smallpox vaccine JYNNEOS® is a requirement for study participation. Prior to enrollment, potential participants will complete a routine adult immunization screening questionnaire (standard of care) and be reviewed by study team for eligibility prior to enrollment.

The entry questionnaires and copy of medical treatment facility (MTF) records and/or Immunization Tracking System documentation will provide baseline demographic and past medical history with specific attention paid to prior immunization history, past medical history of known atopy, HIV disease, coronary artery disease or myocardial infarction, diabetes, carotid artery disease, stroke, or other cardiac disease risk factors such as hyperlipidemia, smoking, hypertension, obesity, etc. All medications being taken prior to enrollment and throughout the study will be documented. The symptom diary/survey will serve as the vehicle to identify symptom and medication changes during study participation. Subjects will use the Defense and Veterans rating scale to rate their pain. The smallpox entry questionnaire, symptom diary and medication log are study-related. A routine screening immunization questionnaire will be completed and the screening form must not have any contraindication identified for the JYNNEOS® vaccine for consideration of enrollment (standard of care). Laboratory tests and ECGs are not standard of care for receiving smallpox vaccination, but are study-related. Participants who develop new onset cardiac symptoms or other symptoms suggestive of possible cardiac involvement which might meet the CDC/DoD case definition of suspect, probable or confirmed myopericarditis, will be referred to Defense Health Agency-Immunization Healthcare Division (DHA-IHD) clinical team as well as cardiology and/or the emergency department for further evaluation. Further ECGs, cardiac biomarkers, cardiac imaging for such participants would be standard of care.

Assigning Subject Identification Numbers:

Upon consent, the subject will be randomly assigned a subject ID number. Subjects requiring concomitant vaccines along with JYNNEOS® will be randomly assigned to one of two groups (1:1 allocation) and that simple computer-generated randomization will be concealed to research staff. The link between subject ID numbers and their names and dates of consent will be maintained at the DHA-IHD research office in a locked filing cabinet within locked offices, and/or in secure, password-protected electronic files, only accessible to the research study team. The linkage document will be destroyed after completion of the final data analysis and IRB acknowledgment/approval of study closure.

Location and Personnel for Study Procedures:

Study procedures will be performed at Defense Health Agency-Immunization Healthcare Division sites or within their AOR by the DHA-IHD study team. (e.g., the Fort Bragg study team may travel to Camp Lejeune to enroll subjects once approved by Camp Lejeune Commanders/Leadership). All study assessments will be performed by members of the investigative team that are specifically designated to perform such activities according to site practices, local laws, and as designated on the appropriate study documents. The Principal Investigator, in collaboration with Associate Investigators and statistician, will be responsible for the interpretation of the statistical information.

Demographic and Medical Data:

Research study staff will collect initial demographics (including age, sex, ethnicity, race, etc.), past medical history, height, weight, and vital signs (such as heart rate, blood pressure, temperature, pulse oximetry and respiratory rate). Demographics will be collected via questionnaire on enrollment day only. Vitals and any updates in medical history will be collected on enrollment day and follow-up visits as well as with weekly email or text surveys (utilizing RedCap or other electronic data management systems as available). Subjects will be asked to provide the study team with information on any medications being used at the time of enrollment and throughout the duration of their participation in the research study. Subjects will be queried on a history of cardiac risk factors and cardiac and atopic disease and be consented for permission to review their medical records, specifically looking for past immunization history, past medical history of cardiac and atopic disease, cardiac risk factors (such as hypertension, smoking, hyperlipidemia, obesity, etc), allergy/atopic history, previous HIV testing, and other relevant medical information. HIV status is specifically queried due to the fact that prelicensure studies identified a higher risk of cardiac events in HIV-positive individuals compared to the general population. Further evaluating for a link between HIV status and the risk of myopericarditis is important in identifying possible risks in this population. The electronic medical records (such as AHLTA, MHS Genesis, CHCS, MEDPROS, ASIMS, MRRS, etc.) may be queried by the research team who may extract subject-specific data to the CRFs/eCRFs.

AQUA Allergy Questionnaire:

Based upon the research team's previous work (unpublished), atopy has been associated with a possible increased risk of vaccinia-induced myopericarditis. Furthermore, in a prelicensure study of JYNNEOSTM patients with atopic dermatitis, the manufacturer reported elevations of troponin in 14.6% of subjects which is significantly elevated above non-atopic patients. Given such, screening for atopy would provide additional information on whether this finding holds true. To this end, the study team will administer a validated instrument (AQUA: Allergy Questionnaire for Athletes) that has the ability to predict atopy with a fairly high degree of sensitivity (58.3%) and specificity (97.1%). \[14\] This questionnaire is preferable to any additional laboratory testing such as total and/or specific IgE and will allow us to avoid additional phlebotomy. The owner of the AQUA questionnaire has provided written permission to use this instrument for the study. The questionnaire will be administered at the initial visit. Additionally, medical history obtained on day of enrollment will ask about a history of hymenoptera hypersensitivity and drug allergies. Such data will be annotated in the CRF/eCRFs. See "other documents" for a copy of the AQUA questionnaire.

Blood Collection:

Subjects will undergo phlebotomy at each of the 5 visits over a 9-week (65 daytime period: once prior to vaccination (at visit 1) and on post-vaccination days 8-14 (visit 2), 28-34 (visit 3), 39-45 (visit 4) and 59-65 (visit 5). All specimens will be labeled with the subject's study identification number (no personal identifiers), type of specimen, date and time of collection, and visit number. Serum/plasma specimens will be processed appropriately and sent to the laboratory for testing. Currently, at the Fort Bragg site, specimen testing will be performed at the WAMC pathology laboratory. Batch processing of specimens for hsTnT may be coordinated with pathology as not to interfere with ongoing clinical care demands. Each stored tube will be tracked with a subject identification number linked database that includes sample type and location in freezer. All specimens will be handled and disposed of in accordance with federal regulations.

This study will use standard clinical blood profiles to determine clinical chemistry and blood markers associated with smallpox vaccination and myopericarditis. Up to \~140 mL (10 tablespoons) of whole blood will be obtained from all subjects via phlebotomy on the following schedule: \~28 mL (2 tablespoons) immediately prior to vaccination (visit 1), \~28 mL (2 tablespoons) between days 8-14 (visit 2), \~28 mL (2 tablespoons) 28-34 days following vaccination # 1 in the series but before vaccination #2 in the series (visit 3), \~28 mL (2 tablespoons) again at day 39-45 (visit 4), and a final draw of \~28 mL (2 tablespoons) at day 59-65 (visit 5).

The following laboratory studies will be conducted at the initial visit once enrolled and prior to vaccination to include standard hematological (CBC with differential) and clinical chemistry markers (Complete Metabolic Profile and Creatinine Phosphokinase) to ensure baseline healthy subjects as well as various myocardial injury markers to include high sensitivity Troponin I (hsTnI), high sensitivity Troponin T (hsTnT) and high sensitivity C-reactive protein (hsCRP). In addition, all follow-up visits will include additional laboratory studies which include the various myocardial injury markers mentioned above to include high sensitivity Troponin I (hsTnI), high sensitivity Troponin T (hsTnT) and high sensitivity C-reactive protein (hsCRP). Primary blood profile endpoints will include mean values of the markers at baseline and correlation between changes in biochemical immunization. Secondary blood profile endpoints will include association with clinical outcomes. Clinical outcomes will include, but not be limited to, referral for evaluation of cardiovascular disease within 30 days post immunization, as well as further analysis to assess possible covariates that may be observed in the initial data analysis.

The research team will conduct all studies using these specimens as research only. However, in the event of an abnormal value identified on baseline labs, the labs will be reviewed by a clinical provider on the research team and the results of the abnormal labs may be relayed to the patient as well as the suggested best course of action for further evaluation (such as scheduling an appointment with the PCM, urgent care or emergency department visit). Labs performed at the local MTF will be entered in CHCS under a research name and number specific to the subject ID and visit number. These lab results will not be reflected in the actual subject's electronic health record under their name, DoD ID, or Social Security number. Every effort will be made to protect confidentiality and no one other than the research team or the Principal Investigator will receive results of the test, with the exceptions noted above for abnormal values. Of note, interpretation of a hsTnT value at a single point in time can be difficult. The diagnostic importance is the change (delta) of the hsTroponin over time. To increase the validity of any significant changes in troponin the research team may batch process specimens so all testing from all 5 visits of an individual subject's specimens are run at the same time (same assay, same machine in same laboratory day) to decrease variability introduced by different assays, different calibrations, lab personnel handling, etc. Though doing so increases sensitivity of detecting significant changes, it also prohibits timely identification of subclinical myopericarditis in a subject. However, given troponin testing is not considered part of the standard of care following any smallpox vaccination and given the clinical significance of an elevated hsTroponin level post-immunization without any accompanying symptoms or ECG changes is not known, and given that the FDA did not require any additional observational studies looking for myocardial injury in JYNNEOSTM vaccinees the investigators feel the risk of such an approach is minimal. Subjects who report clinical symptoms and/or who have abnormal ECG readings would be referred to DHA-IHD clinical teams in consultation with cardiology for further evaluation and management of possible vaccinia-associated myopericarditis (standard of care).

Visit 1/baseline labs Certain laboratory tests will be performed at visit 1 only and will be processed in the local lab or reference labs. These baseline labs tests include a complete blood count (CBC) with differential, comprehensive metabolic profile (CMP), creatinine phosphokinase (CPK), and hsCRP. At the Fort Bragg site, these tests will be performed by WAMC Pathology via the agree

• Secondary Objective [Time frame: Within 35 days following the 2-dose series of a novel, replication-deficient (JYNNEOS™) smallpox vaccine]
• Tertiary Objective [Time frame: Within 35 days following the 2-dose series of a novel, replication-deficient (JYNNEOS™) smallpox vaccine]
• Quaternary Objective [Time frame: Within 35 days following the 2-dose series of a novel, replication-deficient (JYNNEOS™) smallpox vaccine]