Safety and Immunogenicity of LNP-nCOV saRNA-02 Vaccine Against SARS-CoV-2, the Causative Agent of COVID-19

COVAC Uganda is a study that is looking at the use of an innovative self-amplifying RNA (saRNA) vaccine (LNP-nCOV saRNA-02) against the virus (SARS-CoV-2) that causes COVID-19 and assessing the immune response in SARS-CoV-2 antibody seronegative and seropositive individuals. saRNA is designed to amplify the quantity of RNA upon injection to produce further antigen, thereby enabling lower doses for administration. In the trial "COVAC1", Imperial College London is currently evaluating one COVID-19 saRNA vaccine candidate in doses from 0.1-10ug for individuals who are seronegative for SARS-CoV-2 antibodies at baseline. Interim analyses of COVAC1 has shown a dose dependent response; however, up to 50% of seronegative participants receiving doses of 2.5-10ug do not seroconvert. The investigators hypothesize that a lack of seroconversion is due to type I and III interferon (IFN) production, which can inhibit translation and degrade cellular mRNA. Another variable that can enhance antibody production is serological history: recent studies have shown that seropositive individuals respond significantly better than naïve individuals who received the Pfizer or Moderna RNA-based COVID-19 vaccine. Therefore, designing the saRNA backbone to dampen IFN production and evaluating this in individuals seropositive at baseline will inform the optimised use of this innovative technology. In COVAC Uganda, the investigators aim to test an saRNA vaccine modified to dampen the activation of type I and III IFN, to increase antibody production, for individuals who are seronegative and seropositive for SARS-CoV-2 antibodies at baseline, to evaluate whether people with pre-existing seropositivity have enhanced immune responses compared to those without. This trial is NOT looking at whether or not the vaccine is effective in terms of protection. It is just assessing whether and how well the immune system responds based on SARS-CoV-2 antibodies at baseline and its safety.

Background The ongoing pandemic coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in humans in China sometime between October to November 2019, and the disease coronavirus infectious disease 2019 (COVID-19) was identified in China in December 2019.

By the end of January 2021, SARS-CoV-2 had infected with confirmed diagnosis over 106 million people worldwide, resulting in over 2.3 million deaths and over 59 million people recovered from infection. This yields a nominal infection fatality rate (IFR) of \~2%, and around 10% of infected people have been left with health effects lasting for 6 months or more. As of 8 March 2021, Uganda had reported 40,452 coronavirus cases and 334 deaths. Through COVAX, Uganda started rolling out the AstraZeneca vaccine (adenoviral based) from 10 March 2021.

The developing assessment is that the only way the world can exit from the COVID-19 pandemic is through the deployment of an effective vaccine. A number of vaccines have been developed and received emergency use authorization by the United States Food and Drug Administration and/or the World Health Organization. Examples include Pfizer-BioNTech and Moderna's messenger ribonucleic acid (mRNA)-based vaccines which have both shown efficacy at 95%; Oxford-AstraZeneca's ChAdOx1 nCoV-19 and Johnson and Johnson's adenovirus based vaccines which have demonstrated efficacies of 70% and 66% respectively. The problem comes with the scale needed and the time frame in which vaccines need to be developed and deployed. A self-amplifying RNA (saRNA) vaccine provides a novel, feasible, and time-sensitive solution to contribute to addressing the SARS-CoV-2 problem, either during the current pandemic or for ongoing seasonal epidemics.

Studies have demonstrated that nucleic acid-based vaccination can protect against viral infections in non-human primate (NHP) studies, providing proof of concept that gene-based vaccination can induce protective antibodies. However, DNA vaccines require multiple immunizations with the use of electroporation to induce significant immune responses in humans. Non-replicating mRNA-based therapeutics typically require high doses of RNA (100-600 µg). The requirement for high doses, and associated cost, suggest non-replicating mRNA may struggle to produce the potential hundreds of millions of doses required to rapidly respond to a pandemic. In contrast, small animal and non-human primate (NHP) experiments suggest that saRNA induces significantly enhanced responses in comparison to either DNA vaccines delivered with electroporation or mRNA. Indeed, a single immunization with a saRNA vaccine has shown protection against Ebola virus in animal models. Should a ≤10 µg dose of saRNA provide protection from COVID-19, this would provide critical advantages for manufacturing where 100,000 doses can by synthesized in a one liter reaction volume. In contrast to viral vectors, lack of anti-vector immunity provides the opportunity for repeat immunizations with multiple RNA-encoded immunogens.

The first generation saRNA vaccine (LNP-nCoV-saRNA) is already undergoing safety assessment in humans in the COVAC1 trial in the United Kingdom. The safety of this first generation saRNA vaccine has been assessed initially in healthy young adults i.e., 15 participants aged 18-45 years, given one of three different doses (0.1, 0.3 and 1 µg) by injection into the muscle, going slowly from the lowest to the highest over a period of several weeks. To date, there have been no reported serious adverse events (SAEs) associated with this vaccine and a low frequency of Grade 2 events. An additional 35 volunteers have been randomized across each dose. To date, 105 healthy subjects have received two immunizations across each dose, with few Grade 2 events. All participants have safely received both doses. Initial assessment of the first 15 subjects indicates that although the vaccine shows a good safety profile, the levels of seroconversion (75% for the 1ug dose) and binding antibody are lower than anticipated from preclinical models. Indeed, data generated to date suggest responses are at the lower end of a dose response curve. An additional expansion phase has been initiated to explore three higher doses 2.5, 5 and 10ug. The disconnect between human immune response to the vaccine and preclinical models suggests that the level of saRNA expression in humans is less effective than in small animal models. Current data indicate that 5ug dose administered at 0 and 4 weeks induces an acceptable immune response to most (83%) individuals 4-weeks post 2nd vaccination.

The investigators have improved on this initial design (COVAC1) with the modified vector, LNP-nCOV saRNA-02, which is to be investigated within this clinical trial.

This study (COVAC Uganda) will evaluate an innovative saRNA vaccine (LNP-nCOV saRNA-02) designed to increase vaccine potency by shielding the saRNA from cellular proteins known to reduce saRNA expression. The investigators anticipate the safety profile of the modified vaccine to be highly similar to the first generation vaccine already undergoing human trials. COVAC Uganda will use the same assessment criteria as the COVAC1 study designed to see how well the immune system has been activated using different dose levels of the modified vaccine. COVAC Uganda will also enrol SARS-CoV-2 seropositive individuals to evaluate the immune response based on serological history. Seronegative and seropositive participants aged 18-45 will be given one dose of 5.0 ug at 0 weeks and 4 weeks. Seronegative is defined as IgG ≤ 10 AU mL-1 or IgM ≤ 10 AU mL-1, and seropositive is defined as IgG ≥ 10 AU mL-1 or IgM ≥ 10 AU mL-1. The vaccine is given by injection into the muscle of the upper arm. There are likely to be mild side-effects near to the injection site. As observed in COVAC1, there may also be more general side-effects such as headache, temperature and chills. Participants will be asked to record any symptoms in a vaccine diary. In order to see how well the immune system is responding, participants will need to give blood samples several times during the first 6 weeks; then monthly for a few months; then at 6 months.

The investigators predict that the modified vaccine will induce higher levels of neutralising antibodies than the first generation saRNA vaccine. However, if any of the doses do not induce an adequate immune response, there is no known reason why participants who received those doses could not be offered a further booster immunisation with LNP-nCOV saRNA-02 at a dose that has been shown to be safe. Also there is no known reason why participants in COVAC Uganda could not be immunised with other approved SARS-CoV-2 vaccines, including vectored or adjuvanted vaccines, should they be shown to be safe and effective.

Study Rationale This SARS-CoV-2 pandemic has infected over 106 million people and killed over 2.3 million people. As a novel zoonotic virus, no herd immunity is present and the only widely available interventions to date are social distancing to reduce pressure on intensive care beds and health systems, but this is not sustainable for economic reasons. Clinical trials of antivirals and other drug therapies are ongoing but the intervention most likely to mitigate the long-term medical, social and economic impact of the pandemic remains population-wide immunisation.

Despite successfully approved COVID-19 vaccines being rolled out, substantially more candidates are needed to supply 4.265 billion doses for the world's healthcare workers, adults \>65 age, and people at higher risk (with co-morbidities such as diabetes, cardiovascular disease, cancer, obesity or chronic respiratory disease), let alone doses for younger or healthier cohorts.

Recent evidence suggests that immune responses to messenger RNA vaccines is enhanced for individuals previously infected by SARS-CoV-2. This trial investigates the role of seroconversion on the impact of a self-amplifying RNA vaccine candidate, and it will inform on the possible application of LNP-nCOV saRNA-02 as a COVID-19 booster candidate for previously infected individuals.

Pre-clinical data suggest that the LNP-nCOV saRNA-02 vaccine encoding a prefusion stabilised version of the S-glycoprotein will elicit neutralising antibodies in a higher proportion of individuals than natural infection (\<50%), and that the LNP-nCOV saRNA-02 vaccine may provide increased immunogenicity and/or dose reduction over the first generation construct as well as for seropositive individuals.

Study Design This is a phase I clinical study which will build on clinical experience using the LNP nCoV saRNA vaccine currently under evaluation in COVAC1 in the UK. The trial will be conducted in 18-45 year olds in a single centre supervised by the Chief/principal Investigator, by allocating 42 participants into two groups, based on seroconversion status.

Subject Population The study will be conducted in healthy young adults as these individuals generate the most robust responses (18-45 years). Both male and female participants will be included and the trial site will attempt to keep an equal proportion, although the priority will be to ensure timely accrual to the trial.

Dosage and Admiration

Participants will each receive one IM dose of 5 ug of LNP-nCOV saRNA-02, into the deltoid muscle at Week 0 and Week 4 as indicated in the table below:

Study component Serological Status Route Dose Vaccination 1 Vaccination 2 Serological Group SARS-CoV-2 negative antibodies IM 5.0 ug LNP-CoV mod-saRNA-02 Week 0

Visit 2 Week 4

Visit 5 SARS-CoV-2 positive antibodies IM 5.0 ug LNP-CoV mod-saRNA-02 Week 0

Visit 2 Week 4

Visit 5

Safety Evaluations Vaccines are associated with a number of well-characterized local, systemic and laboratory reactions referred to as solicited adverse events. These adverse events will be purposively collected.

Local and systemic assessments will take place on the day of each injection before the injection, and 60 minutes after the injection. Participants should remain at the clinic for at least one hour after each injection.

Participants will be provided with Vaccine diary cards to assist collection and grading of adverse events that start within 7 days of the injection. Study staff will go through the list of solicited adverse events in a structured interview and record these together with grade on the appropriate eCRF in the REDCap database. If any of the events reported at one of the phone visits are moderately severe (grade 2) or worse, participants will be invited to the clinic for review.

Blood (\~10 mL) for routine safety parameters will be collected at all study visits. If the total bilirubin is elevated, study staff will request a result for conjugated bilirubin in order to grade the abnormality and determine any action to be taken with respect to further investigation and interruption to the vaccine schedule.

Vital signs (BP, HR, oxygen saturation and oral temperature) will be measured at every study visit.

Physical examinations of the injection site, and other body systems if indicated, will be performed on the day of each vaccination, and 1 week after. Symptom-directed physical examinations will be performed at all other follow-up visits.

Eligibility Criteria:
Inclusion Criteria:

1. Healthy adults from the following aged 18-45 years on the day of screening
2. At similar risk of acquiring SARS-CoV-2 infection to the general population
3. Willing and able to provide informed consent
4. If female and of childbearing potential, willing to use a highly effective method of contraception from screening until 18 weeks after last injection
5. If male and not sterilised, willing to avoid impregnating female partners from screening until 18 weeks after last injection
6. Willing to avoid all other vaccines from within 4 weeks before the first injection through to 22 weeks after the second injection
7. Willing and able to comply with visit schedule, complete vaccine diaries and provide samples
8. Willing to grant authorised persons access to his/her trial-related medical record and GP records either directly or indirectly

Exclusion Criteria:

1. Pregnant or lactating
2. Has a significant clinical history, physical finding on clinical examination during screening, or presence of a disease that is active or requires treatment to control it, including cardiac, respiratory, endocrine, metabolic, autoimmune, liver, neurological, oncological, psychiatric, immunosuppressive/immunodeficient or other disorders which in the opinion of the investigator is not compatible with healthy status, increases the risk of severe COVID-19, may compromise the volunteer's safety, preclude vaccination or compromise interpretation of the immune response to vaccine. Individuals with mild/moderate, well-controlled comorbidities are allowed.
3. History of anaphylaxis or angioedema
4. Active SARS-CoV-2 infection at enrolment, based on DNA-PCR testing
5. Discordant RDT result
6. History of severe or multiple allergies to drugs or pharmaceutical agents
7. History of severe local or general reaction to vaccination defined as:

   1. local: extensive, indurated redness and swelling involving most of the arm, not resolving within 72 hours
   2. general: fever ≥39.5 °C within 48 hours; bronchospasm; laryngeal edema; collapse; convulsions or encephalopathy within 72 hours
8. Ever received an experimental vaccine against COVID-19
9. Receipt of any immunosuppressive agents within 18 weeks of screening by any route other than topical
10. Detection of antibodies to hepatitis C
11. Detection of antibodies to HIV
12. Grade 1 and above abnormalities in routine laboratory parameters using the FDA toxicity table Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials. https://www.fda.gov/media/73679/download
13. Participating in another clinical trial with an investigational drug or device, or treated with an investigational drug within 28 days of screening.
14. Has received an immunisation within 28 days of screening
15. Has received an authorised COVID-19 vaccine