Background: Mpox is a zoonotic disease caused by the mpox virus (MPXV). It has been endemic in West and Central African countries. However, the soaring number of those has been reported in non-endemic countries since May 2022, making World Health Organization (WHO) declare a global mpox Public Health Emergency of International Concern in July 2022. Those with mpox are primarily young men (96%, and median age of 34 [interquartile range (IQR):29-41 years]), and 84% are self-identified homosexual, bisexual, and men who have sex with men (MSM) . Furthermore, about half of these mpox cases with known human immunodeficiency virus 1(HIV-1, hereafter shown as HIV). WHO recommended prioritizing vaccinating those populations as high-risk populations, including those with HIV, since they will be severely ill if infected mpox virus (MPXV). The smallpox vaccine is expected to offer cross-immunity against MPXV. Under these circumstances, WHO included LC16m8 in the recommended vaccine lists for mpox as the product is expected to have cross-efficacy and immunogenicity against MPXV. Additionally, the safety profile was demonstrated in both adults and children, including infants who have low immuno-functions. Given that Colombia has the fifth highest mpox prevalence worldwide, WHO encouraged the authorities to implement vaccine programs while evaluating the safety and efficacy of LC16m8 as collaborative research. Following WHO initiative, this study is being conducted with the collaboration of various experts from Colombia and Japan on a large scale, with vaccine contributions and funding from Japan and Colombia However, the current infection situation differs from six months ago, and there have been few recent cases of MPXV infection in the country.

Primary objective: To determine the efficacy of the replicating attenuated live vaccinia virus vaccine LC16m8 against laboratory-confirmed mpox and safety in a Colombian population with a high risk of being infected with MPXV(See the Inclusion Criteria), by comparing the immediate vaccination group and the delayed vaccination groups to assess safety and tolerability until 180 days after vaccination. Study design: An open randomized deployment study (1:1 Immediate and Delayed vaccination group).

Study population: People at high risk of serious illness if infected with MPXV and those who engage in risk behaviors for acquiring MPXV infection.

Hypothesis: LC16m8 is a safe and effective vaccine for high-risk immunocompromised populations, including those living with HIV.

Intervention evaluation plan: Vaccinate with LC16m8 those people randomly assigned to two groups: immediate and deferred vaccination 1:1 with a follow-up period of 180 days to evaluate new cases infected by MPXV.

General design: This research is being carried out within the framework of the mpox vaccination implementation program in Colombia. It comprises the following three complementary components:

• Study section 1: Parallel open sequential randomized controlled trial to evaluate the efficacy of LC16m8 vaccine in preventing MPXV infection.
• Study section 2: Cohort study based on the same population base
• Study section 3: Cohort study compared to real world cohort

STUDY SECTION 1: Participants will receive LC16m8 vaccine within 6 weeks (immediate vaccination) or 6 weeks later (delayed vaccination) after randomization. Both groups will be followed up at 14, 30 and 180 days after vaccination, mainly through phone calls.

The study will compare the incidence of new MPXV infections in the early vaccination group from day 14 to day 42 after vaccination with the incidence of rate at which new MPXV infections occur in the delayed vaccination group from 42 days before to 14 days after vaccination; this comparison will be made from the hazard ratio or proportional hazard model.

Additionally, the immune response, including neutralizing antibody titers, will be evaluated from the day of vaccination (Day 1) to 180 days post-vaccination in 60 participants, who will be selected from a single research center and only belonging to the immediate vaccination group. Two 5 mL blood samples (converted to serum) will be collected from the participants for immunogenicity assessments at the following time points: Days 0, 14 (+/-2), 30 (+/-2), and 180 (+/-7) post-vaccination. Another blood sample for immunogenicity assessment will be collected before the vaccine is administered on Day 0.

Safety events will be intensively sought, especially in the first 14 days after vaccine administration and all those arising within 180 days post-vaccination. Participants will also self-report symptoms through a mobile application designed for this purpose.

STUDY SECTION 2 AND 3 (NESTED STUDIES)

- Study of cohorts supported on the same population base: A total of 1,000 vaccinated individuals, including participants in section 1 of the study, will make up the exposed group. The unexposed group will consist of 4,000 unvaccinated individuals from the same population base (a list of HIV patients with a CD4 count greater than 200 cells/ml and PrEP users). who are patients with HIV and CD4 greater than 200 cells/mm3, and PrEP users. The group of unvaccinated individuals are people who meet the study's criteria but did not agree to be vaccinated or withdrew their consent. We will compare the cumulative incidence of these two groups over a 180-day period. We will obtain information about non-participants and withdrawals from the records at the INS and local health entities. We will use the hazard ratio (HR) to compare these groups, and we will also conduct a propensity score analysis.The objective is to demonstrate the effectiveness and safety of the LC16m8 replicating vaccine against laboratory-confirmed MPXV in a Colombian population at high risk of MPXV infection (see inclusion criteria).

Assumptions:

• A relative risk reduction (RRR) of 75% is assumed to calculate the sample size.

• Confidence level: 95%

• Ratio of vaccinated:unvaccinated: 1:4

  • RRR: Risk (Rx) in the unvaccinated group - Rx in the vaccinated group/(Rx) in the unvaccinated group.

The incidence of the event (Mpox case) is considered to be 0.4% per period in the exposed group (I Exp (+)) and 1.6% per period in the unexposed group (I exp (-)), in accordance with the current situation of Mpox infection in Colombia.

Incidence in vaccinated 4/1000 = 0.004 Incidence in unvaccinated 64/4000 = 0.016 RR** = 0.2756 (95% CI: 0.1057 ; 0.7185) **Relative risk (RR): Risk (Rx) in unvaccinated/Rx in vaccinated RAR***: -1.2888 (95% CI: -2.247; - 0.3306) RRR= 0.7244 (95% CI: 0.2815; 0.8943) Power: 0.92 where RR is relative risk, RAR is absolute risk reduction and RRR is relative risk reduction.

The Ho could be rejected.

- Cohort study compared to the real-world cohort: There will be no sample size because all the information from the databases will be used in the cohort, so a sample is not necessary since we will take all the subjects in HIV and PrEP programs.

1. Inclusion Criteria:

   1. Sex: Males and females

   2. Age: ≥18 and ≤ 50 years old

   3. Persons must be willing and sign the Informed Consent (I.C.).

   4. Any of the following conditions including clinical conditions /manifestation:

      • People living with the HIV, with stable infection determined by participant´s being on antiretroviral therapy with a blood CD4+ cell count, ≥ 200 cells/mm3 in the last six months before study enrolment

   5. Persons that use PrEP (HIV Pre-exposure prophylaxis).

   6. Homosexual, Bisexual, or other men who have sex with men (MSM) with multiple sexual partners. Commercial sex workers (CSW) and partners CSW

   7. A female participant is eligible to participate if the participant meets one of the inclusion criteria numbered -1, -2 or -3 above. The participant cannot be pregnant or lactating. Additionally, the female participant must meet one of the following conditions:

      • The participant has non-childbearing potential. To be considered of non-childbearing potential, a female must be post-menopausal for at least one year or surgically sterile. OR
      • The participant has a childbearing potential and agrees to use an effective contraceptive method or abstinence from at least 4 weeks before the vaccine administration until at least 2 months after the administration and have a negative highly sensitive pregnancy test (urine or serum as required by local regulation) within 10 days before vaccination.

2. Exclusion Criteria:

   1. Subjects with an unstable medical condition as determined by their initial medical history, physical examination, or laboratory results
   2. Subjects with a terminal disease.
   3. Subjects with a medical record of anaphylaxis caused by any of the vaccine's excipients or with previous undesired reactions to other vaccines such us (Allergic reactions, Guillain barre syndrome, Varicella zoster, or shingles).
   4. Previous medical record of Mpox.
   5. Subjects living with HIV with a CD4+ T cell count of fewer than 200 cells/mm3.
   6. Pregnant or breastfeeding woman.
   7. Active or medical record of atopic dermatitis or eczema, or with close contact with someone with an active or medical record of atopic dermatitis or eczema.
   8. The presence of a skin condition with extensive breaks in the skin, such as burns, impetigo, contact dermatitis, or zoster (shingles), is not likely to heal by the day of vaccination.
   9. Using immunosuppressive medications, in eye drops, by mouth, or topically (nasal sprays and inhaled corticosteroids are permissible).
   10. Active or past malignancy except for cutaneous basal or squamous cell carcinomas.
   11. An Autoimmune disease.
   12. History of heart failure with decreased left ventricular ejection fraction (<40%).
   13. Medical record of splenectomy.
   14. Medical record of solid organ or bone marrow transplantation.
   15. Medical record of keloid scar development
   16. Psychiatric condition that precludes compliance with the protocol.
   17. People who received or plan to receive licensed live vaccines 30 days before or after study vaccination.
   18. People who received or planned to receive immunoglobulin or other blood products 60 days before HIV screening.
   19. People who received or plan to receive experimental drugs/vaccines 30 days before study vaccination or before study completion.
   20. People who received or planned to receive systemic immunosuppressive therapy and radiation therapy 30 days before or after the study vaccination.
   21. Use of systemic chemotherapy within five years before the study vaccination.
   22. Medical record of smallpox vaccination and/or evidence of scarring at the vaccination site.
   23. Allergies to streptomycin sulfate and/or erythromycin lactobionate.